Monoclonal antibodies for CSPG4 for the diagnosis and treatment of basal breast carcinoma

ABSTRACT

It is disclosed herein that condroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight melanoma associated antigen, is overexpressed on basal breast carcinoma cells (BBC), specifically triple negative basal breast carcinoma cells (TNBC). Methods for detecting basal breast cancer in a subject are disclosed. Methods are also disclosed for inhibiting the growth of a basal breast cancer cell. These methods include contacting the basal breast cancer cell with an effective amount of an antibody that specifically binds CSPG4. Additional treatment methods, and the use of antibody panels, are also described herein.

CROSS REFERENCE TO RELATED APPLICATION

This application is the U.S. National Stage of PCT Application No.PCT/US2009/057578, filed Sep. 18, 2009, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/098,548, filed Sep. 19, 2008, which isincorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant CA16056 andCA 105500 awarded by the National Cancer Institute, National Institutesof Health. The United States government has certain rights in theinvention.

FIELD

This application relates to the field of breast cancer, specifically tomethods for detecting and treating basal breast carcinoma.

BACKGROUND

Breast cancer is the most common type of epithelial cancer among womenin the United States. More than 180,000 women are diagnosed with breastcancer each year. About 1 in 8 women in the United States (approximately12.8 percent) will develop breast cancer during her lifetime. At presentthere are no curative therapies available for breast cancer that hasmetastasized from its site of origination. In addition, there is a needfor diagnostic markers of use in the detection and staging breastcancers.

DNA microarray profiling of breast tumors has identified distinctsubtypes with different clinical outcomes. They include normalbreast-like, Her-2 overexpressing, luminal A and B (predominantly ER⁺),and basal subtypes. Basal-like (referred to as basal) breast cancer(BBC), which includes the A and B subsets, is associated with highgrade, poor prognosis, and younger patients. Because of the lack ofestrogen, progesterone and epidermal growth factor (Her2) receptorexpression, triple negative breast cancer (TNBC), which represents15-20% of all breast cancer, is not suitable for Her2 targeted and/oranti-estrogen-based therapies. Furthermore, BBC is chemo- andradio-resistant. The resistance and aggressive behavior of this tumormay reflect its enrichment for cancer stem cells (CSC) that have thephenotype of CD44⁺ and CD24^(−/lo). CSC are chemo- and radio-resistantand responsible for metastatic spreading and disease recurrence. TNBCcell lines have “stem cell-like” gene expression and are classified asBasal B. A need remains for a method to detect and treat TNBC.

SUMMARY

It is disclosed herein that condroitin sulfate proteoglycan 4 (CSPG4),also known as high molecular weight melanoma associated antigen, isoverexpressed on basal breast carcinoma cells (BBC), of the type alsoknown as triple negative breast cancer cell (TNBC).

Methods for detecting basal breast cancer (BBC) and/or triple negativebreast cancer in a subject are disclosed. These methods includeselecting a subject with breast cancer or suspected of having breastcancer, and detecting the presence of CDPG4 in the sample. In oneexample, the method includes contacting a sample obtained from thesubject with an antibody that specifically binds CSPG4 for a sufficientamount of time to form an immune complex, and detecting the presence ofthe immune complex, wherein the presence of an immune complexdemonstrates the presence of breast cancer in the subject, such as BBCand/or TNBC.

Methods are also disclosed for inhibiting the growth of a basal breastcancer cell and/or a triple negative breast cancer cell. These methodsinclude contacting the basal breast cancer cell and/or triple negativebreast cancer cell with an effective amount of an antibody thatspecifically binds CSPG4, thereby inhibiting the growth of the cancercell. These methods are of use to treat BBC, such as TNBC.

Additional treatment methods, and the use of more than one antibody, arealso described herein.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D are graphs and digital images showing preferential CSPG4expression by TNBC. FIG. 1A is a graph showing an analysis of CSPG4 geneexpression levels in published clinical microarray expression data sets(GSE5460) showed significantly higher levels in estrogen receptor ER⁻,Her²-breast cancers than in ER−/Her2⁺, ER⁺/Her2⁻ and ER⁺/Her2⁺ breastcancers. FIG. 1B is a set of digital images of CSPG4 expression in TNBClesions. Representative IHC staining of human TNBC lesions byCSPG4-specific mAb D2.8.5-C4B8 (3 μg/ml) is shown. The staining wasgraded as A. (−): no CSPG4⁺ cells were detected; B. (+): less than 10%CSPG4⁺ cells were detected; C (++): between 10-80% CSPG4⁺ cells weredetected and D (+++): more than 80% CSPG4⁺ cells were detected. (×200)FIG. 1C is a set of plots showing differential CSPG4 expression andfrequency of CD44⁺/CD24^(−/lo) cells in luminal and basal breastcancer/TNBC cell lines. Cells were sequentially incubated withCSPG4-specific mAb 225.28, PE-labeled anti-mouse IgG antibodies, andFITC-labeled anti-CD24, APC-labeled anti-CD44, and 7-AAD. Stained cellswere subjected to FACS analysis. The percentage of cells stained by mAb225.28 and the mean fluorescence intensity are shown in each histogram.mA low percentage (1.5-13%) of breast cancer cells stained byCSPG4-specific mAb 225.28 is associated with a low percentage (0-34.6%)of CD44⁺/CD24^(−/)lo cells in the three luminal type breast cancer celllines (bottom panel). In contrast, a high percentage (66.7-96.1%) ofbreast cancer cells stained by CSPG4-specific mAb225.28 is associatedwith a high percentage (92.5-99.0%) of CD44⁺/CD24^(−/)lo cells in the 4TNBC cell lines (top panel). FIG. 1D is a set of plots showingenrichment of putative CSC population in CSPG4+ cells in pleuraleffusions from patients with breast carcinoma. 7-AAD⁻/CD45⁻/CSPG4⁺ cellswere analyzed for CD44/CD24 expression. The percentages ofCD44⁺CD24^(−/lo) cells from four patient samples are shown in each dotplot.

FIGS. 2A-2D are digital images and graphs showing the effects ofCSGG4-specific monoclonal antibodies on TNBC cells. FIGS. 2A and 2B area set of digital images and a graph showing the inhibition of TNBC cellgrowth in vitro by CSPG4-specific mAb 225.28. MDA-MB-231 cells weretreated either with mAb 225.28 or control mAb F3C25 in a 3-D (matrigel)setting for 6 days. The PBS, which was used as the solvent for bothmAbs, was used as a reference for 100% cell growth. The pictures weretaken under Zeiss Inverted Fluorescence Microscope (AxioVision Software)of each well (×100) (FIG. 2A) and cells in each well were then harvestedfrom matrigel using Cell Recovery Solution (BD Pharmingen) and countedusing Trypan Blue by two individuals. The results are expressed as %inhibition of cell growth, utilizing the values obtained in PBS only asa reference. The values shown are the mean of three independentexperiments. *** indicates p<0.001 (FIG. 2B). FIG. 2C is a graph showingthe inhibition of TNBC cell adhesion by CSPG4-specific mAb 225.28.MDA-MB-435 cells were seeded and incubated with either CSPG4-specificmAb 225.28, isotype control mAb F3C25 or PBS in an adhesion assay. Theresults are expressed as % inhibition of adhesion, utilizing the valuesobtained in PBS without mAb as a reference. The values shown are themean of three independent experiments. *** indicates p<0.001. FIG. 2D isa graph showing the inhibition of TNBC cell migration by CSPG4-specificmAb 225.28. MDA-MB-231 cells were seeded and incubated with eitherCSPG4-specific mAb 225.28, control mAb F3C25 or PBS in a migrationassay. The results are expressed as % inhibition of migration, utilizingthe values obtained in PBS without mAb as a reference. The values shownare the mean of three independent experiments. *** indicates p<0.001.

FIG. 3 is a digital image showing the down regulation of signalingrelevant to cell growth, adhesion and migration by CSPG4-specific mAb225.28. Western blotting of PKC-α, p-FAK, FAK, p-Erk1/2, Erk1/2, p-Aktand Akt in indicated cultured cells treated with either mAb 225.28,isotype mAb F3C25 or PBS. HLA class I antigens were used as loadingcontrols.

FIGS. 4A-4E are graphs and digital images showing the effects ofCSPG4-specific mAb in vivo. FIGS. 4A and 4B show the inhibition byCSPG4-specific mAb of experimental metastases in vivo. Eight-week oldfemale SCID mice were transplanted intravenously (i.v.) with MDA-MB-231cells (1×10⁶) or MDA-MB-435 cells (2×10⁶) on day 0. On day 3, the micebearing MDA-MB-231 derived metastases were divided into two groups. Onewas treated with CSPG4-specific mAb 225.28 (100 μg/i.v. injection) andone with control mAb F3C25 (100 μg/i.v. injection). The mice bearingMDA-MB-435 derived metastases were divided into three groups. One wastreated with CSPG4-specific mAb 225.28 (100 μg/i.p. injection), one withCSPG4-specific mAb 763.74 (100 μg/i.p. injection), and one with controlmAb (100 μg/i.p. injection). The injections were given twice weekly. Themice transplanted with MDA-MB-231 cells were euthanized on day 79 (FIG.4A) and those transplanted with MDA-MB-435 cells on day 34 (FIG. 4B).Lungs were collected and fixed in Bouin's fixative (Polysciences). Thelung metastases were counted under a dissecting microscope (Zeiss stemiDV4) and analyzed. ** indicates p<0.01, *** indicates p<0.001. FIGS. 4C,4D and 4E are graphs and digital images showing regression byCSPG4-specific mAb of established experimental metastases in vivo.MDA-MB-231 cells (1×10⁶) were injected i.v. to 14 mice on day 0.Subsequently, all tumor bearing mice were randomized into two groups(7/group). Starting on day 20, one group was injected i.v. withCSPG4-specific mAb 225.28 (100 μg/mouse) and the other group wasinjected i.v. with the control mAb F3C25 (100 μg/mouse) every 48 hoursfor a total of three injections. On day 25, all mice were sacrificed andthe lungs were collected and fixed in 10% formalin and paraffin-embeddedfor the following analysis: the sizes/areas of metastatic nodules (inrandomly selected 5 high power fields (×200)/each section) were measuredand calculated by the SPOT IMAGING SOFTWARE Advanced (DiagnosticInstruments, Inc.). The values shown are the mean tumor area of eachgroup. *** indicates p value<0.001 (FIG. 4C); the apoptotic tumor cellsin lung tissue sections were detected by TUNEL assay and quantified bycounting 10 fields/per slide (×200). The values shown are the meanapoptotic tumor cells of each group. ** indicates p<0.01 (FIG. 4D) andthe proliferating tumor cells in lung tissue sections were detected bystaining p-Histone H3 protein and quantified by counting 10 fields/perslide (×200). The values shown are the mean mitotic tumor cells of eachgroup. ** indicates p value<0.01 (FIG. 4E).

FIGS. 5A-5D show the effect of CSPG4-specific mAb in vivo. FIGS. 5A and5B are graphs that show the inhibition by CSPG4-specific mAb ofpost-surgery tumor spontaneous metastases and recurrence in vivo.MDA-MB-435 cells (2×10⁶) were injected into a mammary fat pad of eachSCID mouse on day 0. On day 7, when tumors were measurable, mice weredivided into 3 groups of 5 mice each, such that the mean tumor volume ineach group was similar (16 mm³) Starting on day 7, one of the groups wasinjected i.p. with CSPG4-specific mAb 763.74 (100 μg/mouse) and one withCSPG4-specific mAb 225.28 (100 μg/mouse) twice weekly for a total of 18injections. The third group of mice was injected with a control mAb (100μg/mouse). On day 71, all tumors were removed surgically. The treatmentwith antibodies was continued using the same regimen with 9 additionalinjections. On day 131, all mice were sacrificed, and examined for lungmetastases (FIG. 5A) and local tumor recurrence (FIG. 5B). Thisexperiment was repeated twice. * indicates p value<0.05. FIGS. 5C and 5Dare digital images and a graph showing inhibition of tumor angiogenesisand down regulation of in vivo signaling relevant to cell growth,adhesion and migration by CSPG4-specific mAb. The number of bloodvessels in surgically removed MDA-MB-435 cell-derived primary tumors wasdetected by staining CD31 and quantified using five tumors from eachgroup by capturing 5 random fields (×400) of each section. * indicates pvalue<0.05 (FIGS. 5C and 5D). Western blotting of PKC-α, p-FAK, FAK,p-Erk1/2, Erk1/2, p-Akt and Akt in surgically removed MDA-MB-435cell-derived primary tumors obtained from 5 mice/group treated with mAb225.28 and 5 mice/group treated with the isotype control mAb F3C25 (1tumor sample/per mouse/per lane for both groups) (FIG. 5E). HLA class Iantigens were used as loading controls.

FIGS. 6A-6B are digital images showing the molecular profile of CSPG4expressed by MDA-MB-435 cells. Total RNA was extracted from MDA-MB-435cells. A 439 bp cDNA fragment of CSPG4 (lanes 7 and 8) was synthesizedby RT-PCR (Luo, W., et al., Oncogene 25, 2873-2884, 2006). The PCRproduct was analyzed using 1.5% agarose gel. The melanoma cell linesM14, which does not express CSPG4 (lanes 2 and 6), and M14/CSPG4, whichexpress CSPG4 following transfection with a CSPG4 encoding plasmid DNA(lanes 3 and 7) were used as controls. The housekeeping gene β-actin(lanes 2, 3 and 4) was used as an internal control of RT-PCR. Lanes 1and 5 are DNA molecular markers (FIG. 6A). A lysate from cellsMDA-MB-435 (lanes 3 and 6) was separated by 8% SDS-PAGE for immunoblotanalysis with CSPG4-specific mAb 763.74 (lanes 1-3). The isotype matchedmAb MK2-23 (Kusama et al., J Immunol 143, 3844-3852 (1989) (lanes 4-6)and the M14 (lanes 1 and 4), and M14/CSPG4 cells (lanes 2 and 5) wereused as controls. Calnexin (lanes 1-6), detected by mAb TO-5 (Ogino etal., J Immunol Methods 278, 33-44, 2003) was used as a loading control(FIG. 6B).

DETAILED DESCRIPTION Sequence Listing

The Sequence Listing is submitted as an ASCII text file[8123-81756-07_sequence lisitng.txt, Mar. 16, 2011, 9.93 KB], which isincorporated by reference herein.

In the work described herein, DNA microarray profiles of triple negativebreast cancer (TNBC) followed by immunohistochemistry (IHC) of differentsubtypes of human breast cancer tissues were analyzed to identifyimmunotherapeutic targets. This analysis showed the membrane boundchondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecularweight-melanoma associated antigen (Campoli, M. R., et al., Crit. RevImmunol 24, 267-296, 2004), to be predominantly expressed on TNBC cells.The cell lines used in the studies disclosed herein were defined asbasal by DNA microarray (Neve, R M, et al. (2006), Cancer Cell 10:515-527, incorporated by reference herein).

CSPG4, which is highly conserved through phylogenetic evolution,consists of an N linked 280 kDa glycoprotein and a 450 kDa chondroitinsulfate proteoglycan, both heterogeneous in the expression ofdeterminants on melanoma cells. Because of its high expression withlimited inter- and intra-sesional heterogeneity in at least 80% ofmelanoma lesions and its restricted distribution in normal tissues,CSPG4 has been used as an immunotherapy target in melanoma patients. Itsclinical significance is indicated by the beneficial effect ofCSPG4-specific antibodies induced by CSPG4 mimics (Mittelman et al.,Proc Natl Acad Sci USA 89, 466-470, 1992) on the clinical course of thedisease. CSPG4 and it's rat homolog, NG2 (see, for example, Neve, R. M.,et al., Cancer Cell 10, 515-527, 2006), with its role in cell motilityand migration (see, for example, Burg et al., Exp Cell Res 235, 254-264,1997), is important for tumor cell growth, survival and resistance totherapy. These pathways modulate intergrin function (for example, focaladhesion kinase (FAK)), growth and survival pathways (such as ERk 1, 2and Akt). Furthermore, NG2 has been shown to stimulate the expression offunctional c-Met by an epigenetic mechanism.

Triple negative breast cancer (TNBC) is the most common form of BBC thatis clinically negative for expression of estrogen and progesteronereceptors (ER/PR) and HER2 protein (see Carey, Oncology 22(11),available on the internet on Oct. 1, 2008, incorporated by referenceherein). One characteristic of TNBCs is their ability to migrate andmetastasize (Brabletz et al., Nat Rev Cancer 5, 744-749, 2005), such asto the brain. It is demonstrated herein that the CSPG4 protein isexpressed on TNBC cells. Furthermore, CSPG4 is predominantly expressedon TNBC tissues from patients and highly expressed by a subpopulation ofcells with the CSC phenotype both in TNBC cell lines and in malignantpleural effusions from patients with breast carcinoma.

A functional role of CSPG4 blockade is demonstrated herein. For example,in TNBC xenograft metastasis, it is shown herein that CSPG4-specific mAbsignificantly inhibited growth and caused regression of establishedmetastasis. Without being limiting, results are presented in anexperimental TNBC mouse model and significantly inhibited post-surgerytumor recurrence and lung metastases in an orthotopic TNBC mouse model.The examples describe the use of the TNBC cell lines MDA-MB-435 andMDA-MB-231 used in these two tumor models to generate xenografttumor/metastasis, both of which display the breast CSC phenotype (CD44⁺,CD24^(−/lo)) in 99% of cells. The molecular mechanisms of action ininhibiting tumor recurrence and metastasis mediated by CSPG4-specificmAb involve its ability to inhibit both phosphotidylinositol-3-kinase(PI3K)/PTEN/Akt and MAPK pathways signaling and tumor angiogenesis.Without being bound by theory, the findings are likely to reflect theinhibition of activation of PKC-α, FAK PI3K/Akt and Erk 1, 2 pathways,all of which are associated with the function of CSPG4. These resultsindicate that CSPG4 is an important target to apply antibody-basedimmunotherapy in TNBC.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. An exemplary antigenis CSPG4. The term “antigen” includes all related antigenic epitopes.“Epitope” or “antigenic determinant” refers to a site on an antigen towhich B and/or T cells respond. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least three, andmore usually, at least five or eight to ten amino acids in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are abreast specific antigen, or a prostate specific antigen. Adisease-specific antigen is expressed coincidentally with a diseaseprocess. Specific non-limiting examples of a disease-specific antigenare an antigen whose expression correlates with, or is predictive of,tumor formation, such as breast cancer (for example, BBC or TNBC). Adisease specific antigen may be an antigen recognized by T cells or Bcells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals, including non-human primates. Similarly,the term “subject” includes both human and veterinary subjects.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA,IIIB, and IV).

Breast carcinomas lose the typical histology and architecture of normalbreast glands. Generally, carcinoma cells overgrow the normal cells andlose their ability to differentiate into glandular like structures. Thedegree of loss of differentiation in general is related to theaggressiveness of the tumor. For example, “in situ” carcinoma bydefinition retains the basement membrane intact, whereas as itprogresses to “invasive”, the tumor shows breakout of basementmembranes. Thus one would not expect to see, within breast carcinomas,staining of a discrete layer of basal cells as seen in normal breasttissue. For a discussion of the physiology and histology of normalbreast and breast carcinoma, see Ronnov-Jessen, L., Petersen, O. W. &Bissell, M. J. Cellular changes involved in conversion of normal tomalignant breast: importance of the stromal reaction. Physiol Rev 76,69-125 (1996).

Breast cancers can be divided into groups based on their expressionprofiles. Basal-type carcinomas usually are negative for expression ofestrogen receptor (ER) and negative for expression of HER2 (erbB2) andprogesterone receptor (PR), and thus are referred to as “triple-negativebreast cancers” or “TNBC.” This type of breast cancer is also denotedER⁻/HER2⁻/PR⁻ and represents about 15-20% of all breast cancer, andgenerally cannot be treated using Her2 targeted or estrogen targetedtherapies. It is believed that the aggressive nature of this cancer iscorrelated with an enrichment for cancer stem cells (CSC) with aCD44⁺CD24^(−/lo) phenotype. In some embodiments, basal carcinomas arenegative for expression of progesterone receptor (PR), positive forexpression of epidermal growth factor receptor (EGFR), and positive forexpression of cytokeratin 5 (CK5). This phenotype is denoted as follows:ER⁻/PR⁻/HER2⁻/CK5⁺/EGFR⁺.

The basaluminal subtype of human breast cancer is distinguished fromclassical basal-type tumors in that only a fraction of the cells arepositive for basal cytokeratin 5 (<70%). In addition, 35% of basaluminalbreast cancers display HER2 amplification or overexpression (Laakso etal, 2006, Clin. Cancer Res. 12:4185-4191). In one example, basaluminalsubtype tumors can be characterized as ER⁻/PR⁻/HER2⁺/CK5⁺/EGFR⁺.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treatingbreast and/or prostate cancer. In one embodiment, a chemotherapeuticagent is radioactive compound. One of skill in the art can readilyidentify a chemotherapeutic agent of use (e.g. see Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., ® 2000 Churchill Livingstone,Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy,2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F,Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,Mosby-Year Book, 1993). Combination chemotherapy is the administrationof more than one agent to treat cancer, such as the administration ofantibodies to CSPG4 in combination with a radioactive or chemicalcompound to a subject.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of CSPG4. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; LeuThe term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity or antigenicity.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Degenerate variant: A polynucleotide encoding a CSPG4 polypeptide thatincludes a sequence that is degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences areincluded in this disclosure as long as the amino acid sequence of theCSPG4 polypeptide encoded by the nucleotide sequence is unchanged.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, breast cancer or prostatecancer. Diagnostic methods differ in their sensitivity and specificity.The “sensitivity” of a diagnostic assay is the percentage of diseasedindividuals who test positive (percent of true positives). The“specificity” of a diagnostic assay is one minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis. “Prognostic” is the probability of development (e.g.,severity) of a pathologic condition, such as breast cancer (for example,BBC), or metastasis.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least five or eight to ten amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

Estrogen Receptor (ER): A receptor that is activated by the hormone17β-estradiol (estrogen). The main function of the estrogen receptor isas a DNA binding transcription factor that regulates gene expression.Estrogen receptors are over-expressed in around 70% of breast cancercases, referred to as “ER positive” or “ER⁺.” Therapy for ER⁺ breastcancer involves selective estrogen receptor modulators (SERMS) whichbehave as ER antagonists in breast tissue or aromatase inhibitors. ERstatus is also used to determine sensitivity of breast cancer lesions totamoxifen and aromatase inhibitors.^(L)

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

HER2: Human Epidermal growth factor Receptor 2 (Her2) is also known asHer 2/neu (or ErbB-2, ERBB2). It is a member of the ErbB protein family(also known as the epidermal growth factor receptor family). HER2 hasalso been designated as CD340 (cluster of differentiation 340) and p185.HER2 is notable for its role in the pathogenesis of breast cancer and asa target of treatment. It is a cell membrane surface-bound receptortyrosine kinase and is normally involved in the signal transductionpathways leading to cell growth and differentiation.

Approximately 15-20 percent of breast cancers have an amplification ofthe HER2 gene or overexpression of its protein product. Overexpressionof this receptor in breast cancer has been associated with increaseddisease recurrence and worse prognosis. Because of its prognostic role,breast tumors are routinely checked for overexpression of HER2.Overexpression also occurs in other cancer such as ovarian cancer,stomach cancer, and biologically aggressive forms of uterine cancer,such as uterine serous endometrial carcinoma.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies, such as antibodies that specifically bind CSPG4.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody. The effector molecule can be a toxin. Specific, non-limitingexamples of toxins include, but are not limited to, abrin, ricin,Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40),diphtheria toxin (DT), saporin, restrictocin, or modified toxinsthereof, or other toxic agents that directly or indirectly inhibit cellgrowth or kill cells. For example, PE and DT are highly toxic compoundsthat typically bring about death through liver and heart toxicity inhumans. PE and DT, however, can be modified into a form for use as animmunotoxin by removing the native targeting component of the toxin(e.g., domain Ia of PE and the B chain of DT) and replacing it with adifferent targeting moiety, such as an antibody. A “chimeric molecule”is a targeting moiety, such as a ligand or an antibody, conjugated(coupled) to an effector molecule. The term “conjugated” or “linked”refers to making two polypeptides into one contiguous polypeptidemolecule. In one embodiment, an antibody, such as an antibody thatspecifically binds CSPG4, is joined to an effector molecule (EM). Inanother embodiment, an antibody joined to an effector molecule isfurther joined to a lipid or other molecule to a protein or peptide toincrease its half-life in the body. The linkage can be either bychemical or recombinant means. In one embodiment, the linkage ischemical, wherein a reaction between the antibody moiety and theeffector molecule has produced a covalent bond formed between the twomolecules to form one molecule. A peptide linker (short peptidesequence) can optionally be included between the antibody and theeffector molecule.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen, such as CSPG4, fromwhich the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. MHC binding prediction programs areavailable on the internet, such as ProPed-I, located on the Imtechwebsite (Singh and Raghava, ProPred1: Prediction of promiscuous MHCclass-I binding sites, Bioinformatics, 2003).

Immunogenic composition: A composition comprising an antigenicpolypeptide, such as a CSPG4 polypeptide, that induces a measurableimmune response against cells expressing the polypeptide, such as theCSPG4 polypeptide. The immune response can be a measurable CTL responseagainst cells expressing CSPG4 polypeptide, or a measurable B cellresponse (such as production of antibodies that specifically bind CSPG4)against a CSPG4 polypeptide, such as BBC (TNBC) cells. It further refersto isolated nucleic acids encoding a CSPG4 polypeptide that can be usedto express the CSPG4 polypeptide (and elicit an immune response againstCSPG4). For in vitro use, the immunogenic composition may consist of theisolated protein or peptide. For in vivo use, the immunogeniccomposition will typically comprise the protein or peptide inpharmaceutically acceptable carriers, and/or other agents. Anyparticular peptide, CSPG4 polypeptide, or nucleic acid encoding thepolypeptide, can be readily tested for its ability to induce a CTL or Bcell response by art-recognized assays.

Immunoglobulin (antibody): A protein including one or more polypeptidessubstantially encoded by immunoglobulin genes that specifically binds anantigen. The recognized immunoglobulin genes include the kappa, lambda,alpha (IgA), gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta (IgD), epsilon (IgE)and mu (IgM) constant region genes, as well as the myriad immunoglobulinvariable region genes. Full-length immunoglobulin light chains aregenerally about 25 Kd or 214 amino acids in length. Full-lengthimmunoglobulin heavy chains are generally about 50 Kd or 446 amino acidin length. Light chains are encoded by a variable region gene at theNH2-terminus (about 110 amino acids in length) and a kappa or lambdaconstant region gene at the COOH— terminus. Heavy chains are similarlyencoded by a variable region gene (about 116 amino acids in length) andone of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer thatconsists of two identical pairs of immunoglobulin chains, each pairhaving one light and one heavy chain. In each pair, the light and heavychain variable regions bind to an antigen, and the constant regionsmediate effector functions. Immunoglobulins also exist in a variety ofother forms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies and single chains (e.g., Lanzavecchia etal., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988;Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller andHood, Nature 323:15-16, 1986).

An immunoglobulin light or heavy chain variable region includes aframework region interrupted by three hypervariable regions, also calledcomplementarity determining regions (CDR's) (see, Sequences of Proteinsof Immunological Interest, E. Kabat et al., U.S. Department of Healthand Human Services, 1983). As noted above, the CDRs are primarilyresponsible for binding to an epitope of an antigen. An immune complexis an antibody, such as a monoclonal antibody, chimeric antibody,humanized antibody or human antibody, or functional antibody fragment,specifically bound to the antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody can be joined to human constant segments, such askappa and gamma 1 or gamma 3. In one example, a therapeutic chimericantibody is thus a hybrid protein composed of the variable orantigen-binding domain from a mouse antibody and the constant oreffector domain from a human antibody, although other mammalian speciescan be used, or the variable region can be produced by moleculartechniques. Methods of making chimeric antibodies are well known in theart, e.g., see U.S. Pat. No. 5,807,715.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, i.e., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Exemplary conservative substitutionsare those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr. Humanized immunoglobulins can be constructed bymeans of genetic engineering (e.g., see U.S. Pat. No. 5,585,089).

A human antibody is an antibody wherein the light and heavy chain genesare of human origin. Human antibodies can be generated using methodsknown in the art. Human antibodies can be produced by immortalizing ahuman B cell secreting the antibody of interest. Immortalization can beaccomplished, for example, by EBV infection or by fusing a human B cellwith a myeloma or hybridoma cell to produce a trioma cell. Humanantibodies can also be produced by phage display methods (see, e.g.,Dower et al., PCT Publication No. WO91/17271; McCafferty et al., PCTPublication No. WO92/001047; and Winter, PCT Publication No. WO92/20791,which are herein incorporated by reference), or selected from a humancombinatorial monoclonal antibody library (see the Morphosys website).Human antibodies can also be prepared by using transgenic animalscarrying a human immunoglobulin gene (for example, see Lonberg et al.,PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No.WO91/10741, which are herein incorporated by reference).

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide: A chain of amino acids of between 3 and 30 amino acids inlength, such as from 8 to ten amino acids in length. In one embodiment,a peptide is from about 10 to about 25 amino acids in length. In yetanother embodiment, a peptide is from about 11 to about 20 amino acidsin length. In yet another embodiment, a peptide is about 12 amino acidsin length.

Peptide Modifications: CSPG4 polypeptides include synthetic embodimentsof peptides described herein. In addition, analogues (non-peptideorganic molecules), derivatives (chemically functionalized peptidemolecules obtained starting with the disclosed peptide sequences) andvariants (homologs) of these proteins can be utilized in the methodsdescribed herein. Each polypeptide of this disclosure is comprised of asequence of amino acids, which may be either L- and/or D-amino acids,naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisdisclosure to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a CSPG4 polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealizedthree-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least ten bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is CSPG4polypeptide.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, 15 nucleotides or more inlength. Primers may be annealed to a complementary target DNA strand bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, and then extended along the target DNA strand by aDNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Progesterone receptor (PR): A receptor, also known as NR3C3 (nuclearreceptor subfamily 3, group C, member 3), that is a steroid receptorthat specifically binds progesterone. The progesterone receptor is notexpressed on triple negative basal breast cancer cells.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancers or repressor elements whichcan be located as much as several thousand base pairs from the startsite of transcription. Both constitutive and inducible promoters areincluded (see e.g., Bitter et al., Methods in Enzymology 153:516-544,1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the retrovirus long terminal repeat; theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used.Promoters produced by recombinant DNA or synthetic techniques may alsobe used. A polynucleotide can be inserted into an expression vector thatcontains a promoter sequence which facilitates the efficienttranscription of the inserted genetic sequence of the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific nucleic acid sequences that allowphenotypic selection of the transformed cells

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nucleicacid is one in which the nucleic acid is more enriched than the nucleicacid in its natural environment within a cell. Similarly, a purifiedpeptide preparation is one in which the peptide or protein is moreenriched than the peptide or protein is in its natural environmentwithin a cell. Substantial purification denotes purification from otherproteins or cellular components. In one embodiment, a preparation ispurified (or isolated) such that the protein or peptide represents atleast 50% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%)of the total peptide or protein content of the preparation. The CSPG4polypeptides disclosed herein can be purified (and/or synthesized) byany of the means known in the art (see, e.g., Guide to ProteinPurification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, SanDiego, 1990; and Scopes, Protein Purification: Principles and Practice,Springer Verlag, New York, 1982).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency, will vary depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologues or variants of a CSPG4 polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul et al., Nature Genet. 6:119, 1994, presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a CSPG4 polypeptide are typically characterizedby possession of at least 75%, for example at least 80%, sequenceidentity counted over the full length alignment with the amino acidsequence of CSPG4 using the NCBI Blast 2.0, gapped blastp set to defaultparameters. For comparisons of amino acid sequences of greater thanabout 30 amino acids, the Blast 2 sequences function is employed usingthe default BLOSUM62 matrix set to default parameters (gap existencecost of 11, and a per residue gap cost of 1). When aligning shortpeptides (fewer than around 30 amino acids), the alignment should beperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95%, depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a CSPG4 specific binding agent is an agent thatbinds substantially to a CSPG4 polypeptide. In one embodiment, thespecific binding agent is a monoclonal or polyclonal antibody thatspecifically binds CSPG4.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, often called“helper” T cells, help orchestrate the immune response, includingantibody responses as well as killer T cell responses. CD8⁺ T cellscarry the “cluster of differentiation 8” (CD8) marker. In oneembodiment, a CD8 T cell is a cytotoxic T lymphocytes. In anotherembodiment, a CD8 cell is a suppressor T cell.

Therapeutically active polypeptide: An agent, such as a CSPG4polypeptide that causes induction of an immune response, as measured byclinical response (for example increase in a population of immune cells,production of antibody that specifically binds CSPG4, or measurablereduction of tumor burden). Therapeutically active molecules can also bemade from nucleic acids. Examples of a nucleic acid basedtherapeutically active molecule is a nucleic acid sequence that encodesa CSPG4 polypeptide, wherein the nucleic acid sequence is operablylinked to a control element such as a promoter. Therapeutically activeagents can also include organic or other chemical compounds.Therapeutically active agents can also include adjuvants.

The terms “therapeutically effective fragment of CSPG4” or“therapeutically effective variant of CSPG4” includes any fragment ofCSPG4, or variant of CSPG4, that retains a function of CSPG4, or retainsan antigenic epitope of CSPG4.

In one embodiment, a therapeutically effective amount of a fragment ofCSPG4 is an amount used to generate an immune response, or to treatbreast cancer, specifically BBC, in a subject. Specific, non-limitingexamples are the N-terminal half of CSPG4 or the C-terminal half ofCSPG4. Treatment refers to a therapeutic intervention that ameliorates asign or symptom of breast or prostate cancer, or a reduction in tumorburden.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Monoclonal Antibodies that Bind CSPG4

Antibodies have been produced that specifically bind CSPG4, includingmonoclonal antibodies. In one example, CSPG4 has an amino acid sequenceset forth as:

(SEQ ID NO: 1) EQMREEPEAA YRLIQGPQYG HLLVGGRPTS AFSQFQIDQG EVVFAFTNFS SSHDHFRVLA LARGVNASAV VNVTVRALLH VWAGGPWPQG ATLRLDPTVL DAGELANRTG SVPRFRLLEGPRHGRVVRVP RARTEPGGSQ LVEQFTQQDL EDGRLGLEVG RPEGRAPGPA GDSLTLELWA QGVPPAVASL DFATEPYNAA RPYSVALLSV PEAARTEAGK PESSTPTGEP GPMASSPEPAVAKGGFLSFL EANMFSVIIP MCLVLLLLAL ILPLLFYLRK RNKTGKHDVQ VLTAKPRNGL AGDTETFRKV EPGQAIPLTA  VPGQLFPSee also GENBANK® Accession No. AAI28111 incorporated herein byreference)

CSPG4 is a human melanoma-associated chondroitin sulfate proteoglycanthat plays a role in stabilizing cell-substratum interactions duringearly events of melanoma cell spreading on endothelial basementmembranes. CSPG4 represents an integral membrane chondroitin sulfateproteoglycan expressed by human malignant melanoma cells.

CSPG4 is also known as HMW-MAA. In vivo, it is present in a moleculethat consists of two noncovalently associated glycopolypeptides. One hasan apparent molecular weight of 280K, and the other has an apparentmolecular weight greater than 440K. HMW-MAA is synthesized and expressedby human melanoma cells (Spiro, R. C. et al. F. Biol. Chem. 264:1779(1989); Esko, J. D., et al., Science 241:1092, 1988). Proteoglycans areglycoproteins with glycosaminoglycan (GAG) polysaccharide chainscovalently attached to the serine residue in their core. The CSPG4 coreprotein is initially translated as a precursor with a molecular mass of240K with asparagine N-linked oligosaccharides of the high mannose type.

In another example, the CSPG4 is encoded by the nucleic acid sequenceset forth as:

(SEQ ID NO: 2) gggagcagat gagggaggag ccagaggcag cataccgcct catccaggga ccccagtatg ggcatctcct ggtgggcggg cggcccacct cggccttcag ccaattccag atagaccagggcgaggtggt ctttgccttc accaacttct cctcctctca tgaccacttc agagtcctgg cactggctag gggtgtcaat gcatcagccg tagtgaacgt cactgtgagg gctctgctgcatgtgtgggc aggtgggcca tggccccagg gtgccaccct gcgcctggac cccaccgtcc tagatgctgg cgagctggcc aaccgcacag gcagtgtgcc gcgcttccgc ctcctggagggaccccggca tggccgcgtg gtccgcgtgc cccgagccag gacggagccc gggggcagcc agctggtgga gcagttcact cagcaggacc ttgaggacgg gaggctgggg ctggaggtgggcaggccaga ggggagggcc cccggccccg caggtgacag tctcactctg gagctgtggg cacagggcgt cccgcctgct gtggcctccc tggactttgc cactgagcct tacaatgctgcccggcccta cagcgtggcc ctgctcagtg tccccgaggc agccagagag cagcaccccc acaggcgagc caggccccat ggcatccagc cctgagcccg ctgtggccaa gggaggcttc ctgagcttcc ttgaggccaa catgttcagc gtcatcatccccatgtgcct ggtacttctg ctcctggcgc tcatcctgcc cctgctcttc tacctccgaa aacgcaacaa gacgggcaag catgacgtcc aggtcctgac tgccaagccc cgcaacggcctggctggtga caccgagacc tttcgcaagg tggagccagg ccaggccatc ccgctcacag ctgtgcctgg ccagttattt  cca

See also GENBANK® Accession No. BC128110, incorporated herein byreference. Once of skill in the art can readily use a nucleic acidsequence to produce a polypeptide, such as CSPG4 using standard methodin molecular biology (see, for example, Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

Methods are disclosed herein can be used to detect and treat TNBC,including TNBC in the breast and TNBC that has metastasized to otherorgans. Methods can be designed that utilize nucleic acid techniques todetect the expression of CDPG4, such as PCR-based analysis, such asRT-PCT.

In some embodiments, the methods disclosed herein utilize antibodies,such as monoclonal or polyclonal antibodies, that specifically bindCSPG4 to form an immune complex. Exemplary antibodies of use aredisclosed in PCT Publication No. WO 89/11296, which is incorporated byreference herein. These antibodies include antibodies that bind highmolecular weight melanoma associated antigen (HMW-MAA), as the aminoacid sequence of HMW-MAA is identical to CSPG4.

Exemplary antibodies of use include mouse monoclonal antibodies 225.28;763.74; VF1-TP41.2; VT80.112; 653.25; 763.74; TP61.5 and T8-203 (see PCTPublication No. 89/11296; Drake et al., Cancer Immunol. Immunother. DOI10: 1007, s00262-008-0567-5, 2008; Goto et al., Clin. Cancer Res. 14:3401-3407, 2008, all incorporated by reference herein. Monoclonalantibodies 225.28 and 763.74 were deposited previously by another at theAmerican Type Culture Collection (ATCC). Several hybridomas secretingantibodies that specifically bind CSPG4 were previously deposited byothers in accordance with the Budapest Treaty). In one specific example,the antibody is monoclonal antibody (mAb) 225.28, or is a chimeric,humanized or fully human antibody that specifically binds the epitopebound by mAb 225.28. In a further embodiment, the antibody is afunctional fragment of monoclonal antibody (mAb)) 225.28, a chimeric,humanized or fully human antibody that specifically binds the epitopebound by mAb 225.28. In another specific example, the antibody is mAb763.74, or is a chimeric, humanized or fully human antibody thatspecifically binds the epitope bound by mAb 763.74. Similarly, theantibody can be a functional fragment of mAb 763.74, or a functionalfragment of a chimeric, humanized or fully human antibody thatspecifically binds the epitope bound by mAb 763.74. In some embodiments,the antibodies are any fully human antibody that specifically bindsCSPG4.

Chimeric, humanized and fully human antibodies, and antibody fragments,wherein these antibodies or fragments specifically bind CSPG4, are ofuse in the methods disclosed herein. A panel of different antibodiesthat bind different epitopes, and/or functional fragments of theseantibodies, can also be used in any of the methods disclosed herein.Thus, the disclosed methods can include the use of one, two, three,four, five or six antibodies, or functional fragments thereof, thatspecifically bind CSPG4.

Humanized monoclonal antibodies are produced by transferring donorantibody (from an antibody disclosed herein) complementarity determiningregions from heavy and light variable chains of the mouse immunoglobulininto a human variable domain, and then substituting human residues inthe framework regions of the donor counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of the constantregions of the donor antibody. Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyenet al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci.U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; andSinger et al., J. Immunol. 150:2844, 1993.

In one embodiment, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 65% identical tothe sequence of the donor immunoglobulin heavy chain variable regionframework. Thus, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 75%, at leastabout 85%, at least about 99% or at least about 95%, identical to thesequence of the donor immunoglobulin heavy chain variable regionframework. Human framework regions, and mutations that can be made in ahumanized antibody framework regions, are known in the art (see, forexample, U.S. Pat. No. 5,585,089).

Antibodies include intact molecules as well as fragments thereof, suchas Fab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of specifically binding the epitopicdeterminant. These antibody fragments retain some ability to selectivelybind with their antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. An exemplary scFVthat specifically binds CSPG4 is scFv-Fc C21; a scFV fragment can beused in any of the methods disclosed herein.

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). An epitope is any antigenic determinant onan antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

In one example, the variable region included in the immunotoxin is anFv, which includes the variable region of the light chain and thevariable region of the heavy chain expressed as individual polypeptides.Fv antibodies are typically about 25 kDa and contain a completeantigen-binding site with 3 CDRs per each heavy chain and each lightchain. The V_(H) and the V_(L) can be expressed from two individualnucleic acid constructs. If the V_(H) and the V_(L) are expressednon-contiguously, the chains of the Fv antibody are typically heldtogether by noncovalent interactions. However, these chains tend todissociate upon dilution, so methods have been developed to crosslinkthe chains through glutaraldehyde, intermolecular disulfides, or apeptide linker. Thus, in one example, the Fv can be a disulfidestabilized Fv (dsFv), wherein the heavy chain variable region and thelight chain variable region are chemically linked by disulfide bonds.

One of skill in the art will realize that conservative variants of theantibodies can be produced. Such conservative variants employed in dsFvfragments or in scFv fragments will retain critical amino acid residuesnecessary for correct folding and stabilizing between the V_(H) and theV_(L) regions, and will retain the charge characteristics of theresidues in order to preserve the low pI and low toxicity of themolecules. Amino acid substitutions (such as at most one, at most two,at most three, at most four, or at most five amino acid substitutions)can be made in the V_(H) and the V_(L) regions to increase yield.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5 S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent (Inbar et al., Proc. Nat'lAcad. Sci. U.S.A. 69:2659, 1972). Alternatively, the variable chains canbe linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Thus, a dsFvcan be produced. In an additional example, the Fv fragments compriseV_(H) and V_(L) chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the V_(H) and V_(L) domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra).

In some embodiments, the disclosed method utilize immunoconjugates.Immunoconjugates include, but are not limited to, molecules in whichthere is a covalent linkage of a diagnostic or therapeutic agent with anantibody. A therapeutic agent is an agent with a particular biologicalactivity directed against a particular target molecule or a cell bearinga target molecule. Therapeutic agents include various drugs such asvinblastine, daunomycin and the like, and effector molecules such ascytotoxins such as native or modified Pseudomonas exotoxin or Diphtheriatoxin, encapsulating agents (e.g., liposomes), which themselves containpharmacological compositions, target moieties and ligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell and the biological effect desired. Thus, forexample, the therapeutic agent may be an effector molecule that iscytotoxin which is used to bring about the death of a particular targetcell. Conversely, where it is merely desired to invoke a non-lethalbiological response, a therapeutic agent can be conjugated to anon-lethal pharmacological agent or a liposome containing a non-lethalpharmacological agent.

Toxins can be employed with antibodies that bind the any extracellularportion of a CSPG4 polypeptide and fragments, such as a svFv or a dsFv,to yield chimeric molecules, which are of use as immunotoxins. Exemplarytoxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxinand subunits thereof, ribotoxin, ribonuclease, saporin, andcalicheamicin, as well as botulinum toxins A through F. These toxins arewell known in the art and many are readily available from commercialsources (for example, Sigma Chemical Company, St. Louis, Mo.).

Diphtheria toxin is isolated from Corynebacterium diphtheriae.Typically, diphtheria toxin for use in immunotoxins is mutated to reduceor to eliminate non-specific toxicity. A mutant known as CRM107, whichhas full enzymatic activity but markedly reduced non-specific toxicity,has been known since the 1970's (Laird and Groman, J. Virol. 19:220,1976), and has been used in human clinical trials. See, U.S. Pat. No.5,792,458 and U.S. Pat. No. 5,208,021. As used herein, the term“diphtheria toxin” refers as appropriate to native diphtheria toxin orto diphtheria toxin that retains enzymatic activity but which has beenmodified to reduce non-specific toxicity.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The term“ricin” also references toxic variants thereof. For example, see U.S.Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communisagglutinin (RCA) occurs in two forms designated RCA₆₀ and RCA₁₂₀according to their molecular weights of approximately 65 and 120 kD,respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543,1972). The A chain is responsible for inactivating protein synthesis andkilling cells. The B chain binds ricin to cell-surface galactoseresidues and facilitates transport of the A chain into the cytosol(Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

Ribonucleases have also been conjugated to targeting molecules for useas immunotoxins (see Suzuki et al., Nat Biotech 17:265-270, 1999).Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in,e.g., Rathore et al., Gene 190:31-35, 1997; and Goyal and Batra, Biochem345 Pt 2:247-254, 2000. Calicheamicins were first isolated fromMicromonospora echinospora and are members of the enediyne antitumorantibiotic family that cause double strand breaks in DNA that lead toapoptosis (see, e.g., Lee et al., J. Antibiot 42:1070-1087. 1989). Thedrug is the toxic moiety of an immunotoxin in clinical trials (see,e.g., Gillespie et al., Ann Oncol 11:735-741, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B-chain (abrin-b)binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

In one embodiment, the toxin is Pseudomonas exotoxin (PE). NativePseudomonas exotoxin A (“PE”) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells. The native PE sequenceand the sequence of modified PE are provided in U.S. Pat. No. 5,602,095,incorporated herein by reference. In one embodiment, native PE has asequence set forth as:

(SEQ ID NO: 3)      AEEAFDLWNE CAKACVLDLK DGVRSSRMSV DPAIADTNGQ GVLHYSMVLE GGNDALKLAI DNALSITSDG LTIRLEGGVE PNKPVRYSYT RQARGSWSLN WLVPIGHEKP SNIKVFIHEL NAGNQLSHMS PIYTIEMGDE LLAKLARDAT FFVRAHESNE MQPTLAISHA GVSVVMAQTQ PRREKRWSEW ASGKVLCLLDPLDGVYNYLA QQRCNLDDTW EGKIYRVLAG NPAKHDLDIK PTVISHRLHF PEGGSLAALT AHQACHLPLE TFTRHRQPRG WEQLEQCGYP VQRLVALYLA ARLSWNQVDQ VIRNALASPG SGGDLGEAIR EQPEQARLAL TLAAAESERF VRQGTGNDEA GAANADVVSL TCPVAAGECA GPADSGDALL ERNYPTGAEFLGDGGDVSFS TRGTQNWTVE RLLQAHRQLE ERGYVFVGYH GTFLEAAQSI VFGGVRARSQ DLDAIWRGFY IAGDPALAYG YAQDQEPDAR GRIRNGALLR VYVPRSSLPG FYRTSLTLAA PEAAGEVERL IGHPLPLRLD AITGPEEEGG RLETILGWPL AERTVVIPSA IPTDPRNVGG DLDPSSIPDK EQAISALPDY ASQPGKPPRE DLK

The method of action of PE is inactivation of the ADP-ribosylation ofelongation factor 2 (EF-2). The exotoxin contains three structuraldomains that act in concert to cause cytotoxicity. Domain Ia (aminoacids 1-252) mediates cell binding. Domain II (amino acids 253-364) isresponsible for translocation into the cytosol and domain III (aminoacids 400-613) mediates ADP ribosylation of elongation factor 2. Thefunction of domain Ib (amino acids 365-399) remains undefined, althougha large part of it, amino acids 365-380, can be deleted without loss ofcytotoxicity. See Siegall et al., J. Biol. Chem. 264:14256-14261, 1989.

The term “Pseudomonas exotoxin” (“PE”) as used herein refers asappropriate to a full-length native (naturally occurring) PE or to a PEthat has been modified. Such modifications may include, but are notlimited to, elimination of domain Ia, various amino acid deletions indomains Ib, II and III, single amino acid substitutions and the additionof one or more sequences at the carboxyl terminus, such as KDEL (SEQ IDNO: 4) and REDL (SEQ ID NO: 5) (see Siegall et al., supra). In severalexamples, the cytotoxic fragment of PE retains at least 50%, such asabout 75%, about 90%, or about 95% of the cytotoxicity of native PE. Inone embodiment, the cytotoxic fragment is more toxic than native PE.

Thus, the PE used in the immunotoxins disclosed herein includes thenative sequence, cytotoxic fragments of the native sequence, andconservatively modified variants of native PE and its cytotoxicfragments. Cytotoxic fragments of PE include those which are cytotoxicwith or without subsequent proteolytic or other processing in the targetcell (e.g., as a protein or pre-protein). Cytotoxic fragments of PEknown in the art include PE40, PE38, and PE35.

In several embodiments, the PE has been modified to reduce or eliminatenon-specific cell binding, typically by deleting domain Ia, as taught inU.S. Pat. No. 4,892,827, although this can also be achieved, forexample, by mutating certain residues of domain Ia. U.S. Pat. No.5,512,658, for instance, discloses that a mutated PE in which Domain Iais present but in which the basic residues of domain Ia at positions 57,246, 247, and 249 are replaced with acidic residues (glutamic acid, or“E”) exhibits greatly diminished non-specific cytotoxicity. This mutantform of PE is sometimes referred to as PE4E. PE40 is a truncatedderivative of PE (see, Pai et al., Proc. Nat'l Acad. Sci. U.S.A.88:3358-3362, 1991; and Kondo et al., J. Biol. Chem. 263:9470-9475,1988). PE35 is a 35 kD carboxyl-terminal fragment of PE in which aminoacid residues 1-279 have deleted and the molecule commences with a metat position 280 followed by amino acids 281-364 and 381-613 of nativePE. PE35 and PE40 are disclosed, for example, in U.S. Pat. No. 5,602,095and U.S. Pat. No. 4,892,827.

In some embodiments, the cytotoxic fragment PE38 is employed. PE38 is atruncated PE pro-protein composed of amino acids 253-364 and 381-613 ofSEQ ID NO: 3 which is activated to its cytotoxic form upon processingwithin a cell (see e.g., U.S. Pat. No. 5,608,039, and Pastan et al.,Biochim. Biophys. Acta 1333:C₁-C₆, 1997).

While in some embodiments, the PE is PE4E, PE40, or PE38, any form of PEin which non-specific cytotoxicity has been eliminated or reduced tolevels in which significant toxicity to non-targeted cells does notoccur can be used in the immunotoxins disclosed herein so long as itremains capable of translocation and EF-2 ribosylation in a targetedcell.

Conservatively modified variants of PE or cytotoxic fragments thereofhave at least about 80% sequence identity, such as at least about 85%sequence similarity, at least about 90% sequence identity, or at leastabout 95% sequence similarity at the amino acid level, with the PE ofinterest, such as PE38.

With the antibodies and immunotoxins herein provided, one of skill canreadily construct a variety of clones containing functionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same effector molecule (“EM”) or antibody sequence. Thus,nucleic acids encoding antibodies and conjugates and fusion proteins areprovided herein.

Nucleic acid sequences encoding the immunotoxins can be prepared by anysuitable method including, for example, cloning of appropriate sequencesor by direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., Meth. Enzymol. 68:90-99, 1979; thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979;the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,e.g., using an automated synthesizer as described in, for example,Needham-VanDevanter et al. Nucl. Acids Res. 12:6159-6168, 1984; and, thesolid support method of U.S. Pat. No. 4,458,066. Chemical synthesisproduces a single stranded oligonucleotide. This may be converted intodouble stranded DNA by hybridization with a complementary sequence, orby polymerization with a DNA polymerase using the single strand as atemplate. One of skill would recognize that while chemical synthesis ofDNA is limited to sequences of about 100 bases, longer sequences may beobtained by the ligation of shorter sequences.

In one embodiment, the nucleic acid sequences encoding the immunotoxinare prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Sambrook et al.,supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, an immunotoxin of use is prepared by inserting the cDNAwhich encodes a variable region into a vector which comprises the cDNAencoding the EM. The insertion is made so that the variable region andthe EM are read in frame so that one continuous polypeptide is produced.The polypeptide contains a functional Fv region and a functional EMregion. In one embodiment, cDNA encoding a cytotoxin is ligated to ascFv so that the cytotoxin is located at the carboxyl terminus of thescFv. In one example, cDNA encoding a Pseudomonas exotoxin (“PE”),mutated to eliminate or to reduce non-specific binding, is ligated to ascFv so that the toxin is located at the amino terminus of the scFv. Inanother example, PE38 is located at the amino terminus of the scFv (suchas scFv-Fc C21). In a further example, cDNA encoding a cytotoxin isligated to a heavy chain variable region of an antibody that binds theantigen of interest so that the cytoxin is located at the carboxylterminus of the heavy chain variable region. The heavy chain-variableregion can subsequently be ligated to a light chain variable region ofthe antibody using disulfide bonds. In a yet another example, cDNAencoding a cytotoxin is ligated to a light chain variable region of anantibody that binds the antigen (for example, CSPG4), so that thecytotoxin is located at the carboxyl terminus of the light chainvariable region. The light chain-variable region can subsequently beligated to a heavy chain variable region of the antibody using disulfidebonds.

Once the nucleic acids encoding the immunotoxin is isolated and cloned,the protein can be expressed in a recombinantly engineered cell such asbacteria, plant, yeast, insect and mammalian cells. One or more DNAsequences encoding an immunotoxin can be expressed in vitro by DNAtransfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

Polynucleotide sequences encoding the immunotoxin can be operativelylinked to expression control sequences. An expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences. The expression control sequences include,but are not limited to appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons.

The polynucleotide sequences encoding the immunotoxin can be insertedinto an expression vector including, but not limited to a plasmid, virusor other vehicle that can be manipulated to allow insertion orincorporation of sequences and can be expressed in either prokaryotes oreukaryotes. Hosts can include microbial, yeast, insect and mammalianorganisms. Methods of expressing DNA sequences having eukaryotic orviral sequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the immunotoxin,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982). One of skill in the artcan readily use an expression systems such as plasmids and vectors ofuse in producing proteins in cells including higher eukaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide may becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the recombinantimmunotoxins can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially purecompositions of at least about 90 to 95% homogeneity are disclosedherein, and 98 to 99% or more homogeneity can be used for pharmaceuticalpurposes. Once purified, partially or to homogeneity as desired, if tobe used therapeutically, the polypeptides should be substantially freeof endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (e.g., 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

In addition to recombinant methods, the immunoconjugates, EM, andantibodies disclosed herein can also be constructed in whole or in partusing standard peptide synthesis. Solid phase synthesis of thepolypeptides of less than about 50 amino acids in length can beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, andStewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (e.g., by the use of the coupling reagentN,N′-dicyclohexylcarbodiimide) are well known in the art.

Pharmaceutical Compositions and Therapeutic Methods Using Antibodies

Compositions including an antibody that specifically bind CSPG4, asdisclosed above, can be used for the treatment of basal breast carcinoma(BBC). In one example, methods are disclosed herein for the treatment oftriple negative basal breast cancer (TNBC), which do not expressestrogen, progesterone, or Her2. Thus, compositions comprising atherapeutically effective amount of an antibody that specifically bindsCSPG4 for use in treating BBC, such as TNBC, are provided herein. In oneexample, the BBC (such as a TNBC) is metastatic. In additionalembodiments, method are provided for treating colon, ovarian, breast,prostate, lung or pancreatic cancer.

In one embodiment, the method includes selecting a subject diagnosedwith BBC (such as a TNBC), and administering to the subject atherapeutically effective amount of an antibody that specifically bindsCSPG4. In one example, the method includes selecting a subject withmetastatic BBC, such as a metastatic TNBC.

In further embodiments methods are provided for inhibiting the growthand/or metastasis of a cancer cell. The cancer cell can be a colon,ovarian, breast, prostate, lung or pancreatic cancer cell. The cancercell can be a BBC cell. The cancer cell can be a TNBC cell. The cell canbe in vivo or in vitro.

The antibody can be any antibody that specifically binds CSPG4, such asthose described herein. Thus, the antibody can be, but is not limitedto, a monoclonal antibody, a humanized antibody, a fully human antibody,or a functional fragment of an antibody, provided that the antibodyspecifically binds CSPG4. Combinations of these agents can also beutilized in the disclosed methods.

In some non-limiting examples, the antibody is mAb 225.28, 763.74,TP41.2 or TP61.5, a humanized for thereof or a functional fragmentthereof that specifically binds CSPG4. In another non-limiting examplethe antibody is scFv-Fc C21. These antibodies can be used in alone or inany combination. In some embodiments, the antibody is bound to a toxin.

Compositions including the antibodies disclosed herein can be preparedin unit dosage forms for administration to a subject. The amount andtiming of administration are at the discretion of the treating physicianto achieve the desired purposes. In one example, an antibody orimmunotoxin is formulated for parenteral administration, such asintravenous administration. In other examples, the antibody orimmunotoxin is formulated for systemic or local (such as intra-tumor)administration. These compositions are of use in treating subjects withbreast cancer, such as BBC (for example, TNBC). Thus, the methodsdisclosed herein include selecting a subject with a BBC such as a TNBC.The methods can also include identifying a subject with BBC, and/oridentifying a subject with TNBC, using an antibody that specificallybinds CSPG4, as disclosed below, and then treating the subject. In someexamples, the BBC is metastatic BBC, such as a metastatic TNBC. In someexamples, the method is a method for treating BBC, such as a TNBC in asubject.

The compositions for therapeutic administration will commonly comprise asolution of the antibody, such as an immunotoxin, dissolved in apharmaceutically acceptable carrier, such as an aqueous carrier, foradministration to a subject with breast cancer, such as BBC (forexample, TNBC). A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well-known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of fusion protein in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the patient's needs.

The antibodies, such as can be administered to slow or inhibit thegrowth of cells that express the antigen (CSPG4), such as BBC cells, forexample TNBC cells. In these applications, a therapeutically effectiveamount of an antibody that binds CSPG4, for example an immunotoxin, isadministered to a subject in an amount sufficient to inhibit growth ofantigen-expressing cells, such as BBC (TNBC) cells. Suitable subjectsinclude those with a breast cancer that expresses a CSPG4, such as asubject with BBC, for example TNBC. Suitable subjects also include thosewith a metastatic cancer thought to be BBC, and/or with a metastaticcancer thought to be TNBC.

A typical pharmaceutical of an antibody, such as an immunotoxincomposition, for intravenous administration includes about 0.1 to 10 mgper patient per day. Dosages from 0.1 up to about 100 mg per patient perday may be used, particularly if the agent is administered to a secludedsite and not into the circulatory or lymph system, such as into a bodycavity or into a lumen of an organ. Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 19th ed., Mack Publishing Company,Easton, Pa. (1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% Sodium Chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of Rituxan® in 1997. Antibody drugs can beadministered by slow infusion, rather than in an IV push or bolus. Inone example, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In any event, the composition should provide a sufficientquantity of at least one of the antibodies disclosed herein toeffectively treat the patient with breast cancer (e.g. BBC). The dosagecan be administered once but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. A therapeuticallyeffective amount of the antibody is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Thesecompositions can be administered in conjunction with anotherchemotherapeutic agent, either simultaneously or sequentially.

Controlled release parenteral formulations of the compositions includingthe antibody can be made as implants, oily injections, or as particulatesystems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa.,(1995) incorporated herein by reference. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, e.g., Kreuter, J., Colloidal Drug DeliverySystems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp.219-342, 1994; and Tice & Tabibi, Treatise on Controlled Drug Delivery,A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339,1992, both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibody orimmunoconjugate compositions disclosed herein. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). Forexample, the block copolymer, poloxamer 407, exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known. See, e.g.,U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496, each of which is incorporated herein by reference.

A therapeutically effective amount of an antibody will depend upon theseverity of the disease and the general state of the patient's health. Atherapeutically effective amount of the antibody is that which provideseither subjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

In some embodiments, the antibody that specifically binds CSPG4 isadministered with other agents, such as chemotherapeutic agents, eithersimultaneously or sequentially. Many chemotherapeutic agents arepresently known in the art. Thus, the method can include administering atherapeutically effective amount of an antibody that specifically bindsCSPG4 and a therapeutically effective amount of a chemotherapeuticagent. In several embodiments, the chemotherapeutic agents is selectedfrom the group consisting of mitotic inhibitors, alkylating agents,anti-metabolites, intercalating antibiotics, growth factor inhibitors,cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survivalagents, biological response modifiers, anti-hormones, e.g.anti-androgens, and anti-angiogenesis agents.

Examples of alkylating agents include nitrogen mustards (such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine. Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocorticalsuppressants (such as mitotane and aminoglutethimide). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and megestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testosterone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsthat can be concurrently administered with the disclosed immunotherapyinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor; Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech).

In several specific non-liming examples, a therapeutically effectiveamount of ABRAXANE® (paclitaxel protein-bound particles for injectablesuspension), ADRIAMYCIN® (doxorubicin), AREDIA® (generic name,pamidronate disodium), ARIMIDEX® (anastrozole), AROMASIN® (exemestane),CYTOXAN® (cyclophosphamide), ELLENCE® (epirubicin), EVISTA®(raloxifene), FARESTON® (toremifene), FEMARA® (letrozole), HERCEPTIN®(trastuzumab), MEGACE® (megestrol), Tamoxifen, TAXOL® (paclitaxel),TAXOTERE® (docetaxel), XELODA® (capecitabine), ZOLADEX® (goserelinacetate), and/or ZOMETA® (generic name, zoledronic acid) isadministered.

Diagnosis of Basal Breast Carcinoma

It is disclosed herein that CSPG4 is differentially expressed in humanbreast cancers. Specifically CSPG4 is expressed in BBC (specificallyTNBC), which is often chemo- and radio-resistant. CSPG4 is expressed incancer stem cells (CSC). Thus, expression of a CSPG4 polypeptide can beused to diagnose BBC (TNBC), can be used to stage breast cancer, or canbe used to determine the prognosis of a subject with breast cancer, suchas BBC, for example TNBC. Thus, the use of an antibody that specificallybinds CSPG4 for the diagnosis of BBC, such as TNBC, is disclosed herein.In some embodiments, the method determines if the subject has BBC, suchas a TNBC, as compared to another form of breast cancer. The method ofdiagnosis can be used to determine if a metastasis, such as a brainmetastasis, is a BBC, such as a TNBC.

A method is provided herein for the detection of the expression of aCSPG4 polypeptide in cells or tissue by analyzing expression of thepolypeptide in a biological sample. The sample can be any sample,including, but not limited to, tissue from biopsies, autopsies, andpathology specimens. Biological samples also include sections oftissues, such as frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum, orurine. In one example, the biological sample is a breast tissue sample.In another embodiment, the biological sample is a sample of a tissuefrom another organ (not the breast), so that the presence of metastaticbreast cancer is determined, such as in a brain biopsy. The biologicalsample is typically obtained from a mammal, such as a rat, mouse, cow,dog, guinea pig, rabbit, or primate, such as a human.

In a further embodiment, the subject has cancer, or is suspected ofhaving cancer, such as breast cancer. In one example, the breast canceris BBC, such as TNBC.

A method is also provided herein for detecting CSPG4 in a biologicalsample. The method includes contacting the sample with one or more of anantibody that specifically binds CSPG4 to form an antibody-CSPG4complex. The presence or absence of the complex is detected. The methodsare of use to improve the confidence of a tissue diagnosis, such as BBC(for example, TNBC), such as to confirm a diagnosis, or to determine theorigin of a tumor. Thus, the method disclosed herein can be used toconfirm the diagnosis of the BBC, such as TNBC.

The methods can include selecting a subject in need of diagnosis, suchas a subject with a breast cancer or suspected of having breast cancer(such as a subject identified using mamography), and obtaining a samplefrom this subject. In several examples, the methods include selecting asubject with breast cancer, and using the methods disclosed herein todetermine if the breast cancer is a BBC, such as a TNBC.

Antibodies that specifically bind CSPG4 are used in the methodsdisclosed herein. Antibodies include polyclonal and monoclonalantibodies, humanized and chimeric antibodies, as well as fully humanantibodies. In some embodiments, an antibody fragment, wherein theantibody fragment specifically binds CSPG4 is utilized in these methods.In one example, the antibody fragment is an Fv fragment. In a furtherembodiment, the antibody is labeled (such as with a fluorescent,radioactive, or an enzymatic label). In additional examples, theantibodies can be conjugated to compounds including, but not limited to,enzymes, magnetic beads, colloidal magnetic beads, haptens,fluorochromes, metal compounds or radioactive compounds. Methods forlabeling antibodies are well known in the art.

Methods of determining the presence or absence of a protein are wellknown in the art. Assays of use include, but are not limited to,radioimmunoassays (RIAs), enzyme linked immunosorbant assays (ELISA), orimmunohistochemical assays. The method for detecting CSPG4 in abiological sample generally includes the steps of contacting thebiological sample with an antibody which specifically reacts, underimmunologically reactive conditions, to CSPG4. The antibody is allowedto specifically bind under immunologically reactive conditions to forman immune complex, and the presence of the immune complex (boundantibody) is detected directly or indirectly. A control cell, such as anon-transformed cell or section of the same tissue type, can be includedas a control.

In one embodiment, a method is provided for detecting a polypeptide in asample. Kits for detecting a polypeptide will typically comprise anantibody that specifically binds the CSPG4. The antibody can be amonoclonal antibody, a chimeric antibody, a humanized antibody, or ahuman antibody that specifically binds CSPG4. In some embodiments, anantibody fragment, such as an Fv fragment is included in the kit. For invivo uses, the antibody can be a scFv fragment or a human antibody. In afurther embodiment, the antibody is directly labeled (e.g. fluorescent,radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds CSPG4 (e.g. fordetection of BBC, such as TNBC, cells expressing CSPG4). Theinstructional materials may be written, in an electronic form (e.g.computer diskette or compact disk) or may be visual (e.g. video files).The kits may also include additional components to facilitate theparticular application for which the kit is designed. Thus, for example,the kit may additionally contain means of detecting a label (e.g. enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a secondary antibody, orthe like). The kits may additionally include buffers and other reagentsroutinely used for the practice of a particular method. Such kits andappropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay, such asan immunoassay to detect or stage a BBC, such as a TNBC. Although thedetails of the immunoassays may vary with the particular formatemployed, the method of detecting CSPG4 in a biological sample generallycomprises the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, tothe CSPG4 polypeptide. The antibody is allowed to specifically bindunder immunologically reactive conditions to form an immune complex, andthe presence of the immune complex (bound antibody) is detected directlyor indirectly.

The methods also include detecting a nucleic acid in a sample, whereinthe nucleic acid encodes CSPG4. Suitable methods are well known in theart. For example, samples from a subject can be tested to determinewhether nucleic acids encoding CSPG4 are present. In one embodiment, anamplification procedure is utilized to detect nucleic acids encodingCSPG4, such as reverse transcriptase polymerase chain reaction (RT-PCR).In another embodiment, a blotting procedure (e.g. Northern Blot or DotBlot) is used to detect the presence of nucleic acids encoding CSPG4.Thus, probes or primers that specifically hybridize to nucleic acidsencoding CSPG4 are used to detect the presence of a BBC, such as a TNBC.

In an alternative set of embodiments, kits can be provided for detectingnucleic acids encoding CSPG4 in a biological sample. For example, asample from a subject can be tested to determine whether nucleic acidsencoding CSPG4 polypeptide are present, such as mRNA encoding a CSPG4polypeptide. In one embodiment, an amplification procedure is utilizedto detect nucleic acids encoding CSPG4. In another embodiment, ablotting procedure (e.g. Northern Blot or Dot Blot) is used to detectthe presence of nucleic acids. In a further embodiment, nucleic acids ofinterest are amplified, and these nucleic acids are sequenced todetermine if a CSPG4 polypeptide is expressed. Thus, a kit can includeprobes or primers that specifically hybridize under stringentconditions, or highly stringent conditions, to nucleic acids encodingCSPG4.

In one embodiment, a kit provides a primer that amplifies nucleic acidencoding CSPG4. Conveniently, the amplification is performed bypolymerase chain reaction (PCR). A number of other techniques are,however, known in the art and are contemplated for use. For example,Marshall, U.S. Pat. No. 5,686,272, discloses the amplification of RNAsequences using ligase chain reaction, or “LCR,” (Landegren et al.,Science 241:1077, 1988); Wu et al., Genomics 4:569, 1989; Barany, in PCRMethods and Applications 1:5, 1991); and Barany, Proc. Natl. Acad. Sci.U.S.A. 88:189, 1991). Or, the RNA can be reverse transcribed into DNAand then amplified by LCR, PCR, or other methods. An exemplary protocolfor conducting reverse transcription of RNA is taught in U.S. Pat. No.5,705,365. Selection of appropriate primers and PCR protocols aretaught, for example, in Innis et al., eds., PCR Protocols, 1990(Academic Press, San Diego, Calif.). The resultant nucleic acids can besequenced, or can be identified using specific CSPG4 probes usinghybridization and blotting techniques well known in the art.

In one embodiment, the kit includes instructional materials disclosingmeans of use for the primer or probe. The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. The kits may additionally include buffers and otherreagents routinely used for the practice of a particular method. Suchkits and appropriate contents are well known to those of skill in theart.

Use of CSPG4 to Induce an Immune Response to Basal Breast Carcinoma

In one embodiment, methods are provided for producing an immune responseto a CSPG4 polypeptide. The method is of use, for example, for thetreatment of cancer, such as by reducing the growth of the cancer orreducing a sign or a symptom of the cancer in a subject. The cancer canbe a breast cancer, such as a BBC, for example TNBC. The method includesadministering to a subject a therapeutically effective amount of apolypeptide including the CSPG4 polypeptide, or a fragment thereof. Themethod can also include administering to a subject a nucleic acidencoding this polypeptide.

In one embodiment, the method includes administering a therapeuticallyeffective amount of CSPG4 polypeptide in a pharmacologically acceptablecarrier. In additional embodiment, the method includes administering toa subject an isolated immunogenic epitope of CSPG4. The immunogenicepitope can induce a B cell response and/or a T cell response. The useof nucleic acids encoding these polypeptides and epitopes for thetreatment of basal breast cancer are also envisioned. The use of such anepitope can result in the production of antibodies and/or can result ina T cell response, such as the activation of T cells or the productionof cytokines.

The presentation of peptides by MHC Class I molecules involves bindingto the cleft in an MHC Class I molecule through the anchor residues ofthe peptide and ultimate presentation on the cell surface. Dependingupon the particular anchor residues, among other things, certainpeptides may bind more tightly to particular HLA molecules than others.Peptides that bind well are usually “dominant” epitopes, while thosethat bind less well are often “subdominant” or “cryptic” epitopes.Dominant epitopes of either self proteins or foreign proteins evokestrong tolerance or immune responses. Subdominant or cryptic epitopesgenerate weak responses or no responses at all. Without being bound bytheory, tighter binding by dominant epitopes to HLA molecules results intheir denser presentation on the cell surface, greater opportunity toreact with immune cells and greater likelihood of eliciting an immuneresponse or tolerance. MHC Class I molecules present epitopes fromendogenous proteins for presentation to CTL cells. HLA A, HLA B and HLAC molecules bind peptides of about 8 to 10 amino acids in length thathave particular anchoring residues. The anchoring residues recognized byan HLA Class I molecule depend upon the particular allelic form of theHLA molecule. A CD8+ T cell bears T cell receptors that recognize aspecific epitope when presented by a particular HLA molecule on a cell.When a CTL precursor that has been stimulated by an antigen presentingcell to become a cytotoxic T lymphocyte contacts a cell that bears suchan HLA-peptide complex, the CTL forms a conjugate with the cell anddestroys it. In one example, the polypeptides disclosed herein bind andare presented by HLA-A2.1. Using the amino acid sequences for CSPG4, oneof skill in the art can use a computer program to identify epitopes thatwill stimulate T cells. Such programs are publicly available on theinternet, for example ProPed-I, which is available at the Imtechwebsite. Fusion proteins including CSPG4 polypeptides, or immunogenicepitopes thereof, can also be utilized. For example, the CSPG4polypeptide can be fused to an immunoglobulin polypeptide,glutathione-S-transferase, poly-histidine, or beta-galactosidase.

The subject can be any subject of interest. In one embodiment, thesubject has breast cancer, specifically BBC, such as TNBC. Additionalagents can be included, such as, but not limited to, chemotherapeuticagents or adjuvants. The method can include identifying and or selectinga subject with a BBC, such as a TNBC. In one embodiment, the CSPG4polypeptide, or fragment or epitope thereof, is mixed with an adjuvantcontaining two or more of a stabilizing detergent, a micelle-formingagent, and an oil. Suitable stabilizing detergents, micelle-formingagents, and oils are detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No.5,709,860; U.S. Pat. No. 5,270,202; and U.S. Pat. No. 5,695,770, all ofwhich are incorporated by reference. A stabilizing detergent is anydetergent that allows the components of the emulsion to remain as astable emulsion. Such detergents include polysorbate, 80 (TWEEN™)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,Zwittergent™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977; and Hunter, et al. J. Immunol.129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of suchagents are well known in the art. In one embodiment, the agent is chosento have a hydrophile-lipophile balance (HLB) of between 0 and 2, asdefined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent canbe provided in an effective amount, for example between 0.5 and 10%,such as in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, i.e., to provide a vehicle forthe desired antigen, and can have a melting temperature of less than 65°C. such that emulsion is formed either at room temperature (about 20° C.to 25° C.), or once the temperature of the emulsion is brought down toroom temperature. Examples of such oils include squalene, Squalane,EICOSANE™, tetratetracontane, glycerol, and peanut oil or othervegetable oils. In one specific, non-limiting example, the oil isprovided in an amount between 1 and 10%, such as between 2.5 and 5%. Theoil should be both biodegradable and biocompatible so that the body canbreak down the oil over time, and so that no adverse affects, such asgranulomas, are evident upon use of the oil.

An adjuvant can be included in the composition. In one embodiment, theadjuvant is a mixture of stabilizing detergents, micelle-forming agent,and oil available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.). To extend the time during which the peptide or proteinis available to stimulate a response, the peptide or protein can beprovided as an implant, an oily injection, or as a particulate system.The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle (see, e.g., Banga,supra). A particulate carrier based on a synthetic polymer has beenshown to act as an adjuvant to enhance the immune response, in additionto providing a controlled release. Aluminum salts may also be used asadjuvants to produce a humoral immune response. Thus, in one embodiment,the polypeptide is administered in a manner to induce a humoralresponse.

A CSPG4 polypeptide, fragment thereof, or immunogenic epitope, ornucleic acid endocing these polypeptides can be administered by anymeans known to one of skill in the art (see Banga, A., “ParenteralControlled Delivery of Therapeutic Peptides and Proteins,” inTherapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995) such as by intramuscular, subcutaneous,intratumor, or intravenous injection, but even oral, nasal, or analadministration is contemplated. In one embodiment, administration is bysubcutaneous or intramuscular injection.

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding the polypeptide or immunogenic fragment thereof. Atherapeutically effective amount of the polynucleotide can beadministered to a subject in order to generate an immune response. Inone specific, non-limiting example a therapeutically effective amount ofthe polynucleotide is administered to a subject to treat cancer, such asovarian, colon, prostate, breast, lung, or pancreatic cancer.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence can encode CSPG4. In oneembodiment, the method includes administering to a subject atherapeutically effective amount of a nucleic acid encoding CSPG4 in apharmacologically acceptable carrier. More than one nucleic acidsequence encoding a polypeptide can be included in the expressionvector, so that combinations of polypeptides can be administered.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response) and U.S. Pat. No.5,593,972 and U.S. Pat. No. 5,817,637 (which describe operably linking anucleic acid sequence encoding an antigen to regulatory sequencesenabling expression). U.S. Pat. No. 5,880,103 describes several methodsof delivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, apolypeptide can also be expressed by attenuated viral hosts or vectorsor bacterial vectors. Recombinant vaccinia virus, adeno-associated virus(AAV), herpesvirus, retrovirus, or other viral vectors can be used toexpress the peptide or protein, thereby eliciting a CTL response. Forexample, vaccinia vectors and methods useful in immunization protocolsare described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin)provides another vector for expression of the peptides (see Stover,Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a polypeptide is introduceddirectly into cells. For example, the nucleic acid may be loaded ontogold microspheres by standard methods and introduced into the skin by adevice such as Bio-Rad's Helios™ Gene Gun. The nucleic acids can be“naked,” consisting of plasmids under control of a strong promoter.Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites, including tissues in proximity tometastases. Dosages for injection are usually around 0.5 μg/kg to about50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see,e.g., U.S. Pat. No. 5,589,466).

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

No effective targeted treatment is available for triple negative basalbreast cancer (TNBC). Here it is demonstrated that chondroitin sulfateproteoglycan 4 (CSPG4), is predominantly expressed on TNBC cancertissues and overexpressed on TNBC cells and on breast cancer stem cells(referred to as CSC), identified as CD44⁺, CD24^(−/lo) cells, in pleuraleffusions from breast cancer patients and in TNBC cell lines.CSPG4-specific monoclonal antibody (mAb), on its own, significantlyinhibited growth and caused regression of established metastasis in anexperimental TNBC mouse model and significantly inhibited post-surgeryspontaneous lung metastases and tumor recurrence in an orthotopic TNBCmouse model. The human TNBC cell lines used in these two types of tumormodels to generate xenograft tumor/metastasis were MDA-MB-435 andMDA-MB-231, both of which display the breast CSC phenotype (CD44⁺,CD24^(−/lo)) in 99% of cells. Mechanistic studies of the targetedtherapy revealed that the CSPG4-specific monoclonal antibody (mAb) wasable to inhibit both (Phophatidylinositde-3-Kinase) PI3K/PTEN/Akt andMitogen-activated protein (MAP) kinases (MAPK) pathways signaling. Inaddition, the CSPG4-specific mAb was capable of reducing tumorangiogenesis. These findings establish CSPG4 as an important therapeutictarget for antibody based immunotherapy for reducing post-surgery tumorrecurrence and metastasis risk.

Example 1 Materials and Methods

Patients, Pleural Effusions and Cell Preparation.

Pleural effusions from breast cancer patients were obtained under anapproved cell and tissue procurement protocol. Briefly, effusions weredrained using a PleurX catheter and bottles to collect the fluid. Tumorcells were isolated by ficolling the fluid (typically 500 mL-2 L) toremove debris and red blood cell contamination and cryopreserved.Typical yields of cells were 2×10⁸ to 3×10¹¹ cells per liter of fluid.

Mice.

Female SCID/BALB/c mice (C.B-Igh-1^(b)IcrTac-Prkdcscid, 6-8 weeks old)were purchased from NCI.

Cell Lines.

The melanoma and the breast cancer cell lines HS578T, MCF-7, MDA-MB-231,MDA-MB-435s, SK-BR-3, SUM149 and T47D were obtained from DukeComprehensive Cancer Center Cell Culture. All cell lines and humanmelanoma cell lines M14 and M14/CSPG4 transfectant (additional G418 400μg/ml) were maintained in RPMI 1640 medium supplemented with 10% FCS,pen-strep-glutamine (Invitrogen).

Antibodies.

The mAb 225.28, 763.74, TP41.2 and TP61.5 which recognize distinctdeterminants of CSPG4 (Campoli, et al., Crit. Rev Immunol 24, 267-296,2004), the mAb D2.8.5-C4B8 which recognizes a determinant of CSPG4 informalin fixed and paraffin embedded tissue sections, the anti-idiotypicmAb F3C25 (Perosa, & Ferrone, Hum Immunol 23, 255-269, 1988) and MK2-23and the calnexin-specific mAb TO-5, were developed as described (Kusamaet al., J Immunol 143, 3844-3852, 1989); Ogino et al., J Immunol Methods278, 33-44, 2003). All mAb are IgG1, except mAb 225.28 and F3C25, whichare IgG2a; mAb were purified from ascitis by sequential ammonium sulfateand caprylic acid precipitation (Temponi et al, Hybridoma 8, 85-95,1989). PE-anti-mouse IgG, FITC-CD24, APC-CD44, PerCP-CD45, 7-AAD, andlineage cocktail (CD2, 3, 10, 16, 18, 31, 45, 64, 140b) conjugatedantibodies, unconjugated anti-mouse CD31 (PECAM1) antibody werepurchased from BD/Pharmingen. Specific antibodies were purchasedcommercially for: PKCα (Sigma); phosphorylated and non-phopho-FAK (BDBioscience); phosphorylated and pan Akt, phospho-PTEN(Ser380/Thr382/383), phospho-Met (Tyr1234/1235) (D26), phospho-Gab (Tyr307), phospho-Met (Tyr1003), phospho-p44/42 MAPK (Erk1/2),(Thr202/Tyr204) and phopho-Histone H3(Ser10) (Cell signalingtechnology).

Gene Expression Analysis.

RMA gene expression data was obtained for a publicly available,clinically annotated breast cancer data set (GEO accession number:GSE5460) consisting of 125 samples and categorized according to hormonalstatus (ER−/Her2−, ER−/Her2+, ER+/Her2− and ER+/Her2+) (Lu, X., et al.,Breast Cancer Res Treat 108, 191-201, 2008). Gene expression levelcorresponding to chondroitin sulfate proteoglycan 4 (CSPG4)(204736_s_at, 214297_at) was obtained for each subgroup and differencesbetween the groups were analyzed via the non-parametric Mann-Whitney Utest using Graph Pad Prism Software, version 4.03. A two-sided p-valueless than 0.05 was considered statistically significant.

Flow Cytometry Analysis.

Tumor cell preparations from plural effusion were labeled withantibodies to well-characterized surface cell markers (CD44, CD24) toassess the presence and percentage of various cell populations by flowcytometry, looking in particular for CD44⁺CD24^(−/low) cells, as perAl-Hajj et al. to identify CSC. Briefly, cells were stained withCSPG4-specific mAb for 30 mM, washed twice with PBS, and incubated for30 min with PE-labeled anti-mouse IgG antibody. After 3 washes, cellswere stained with FITC-CD24, APC-CD44 and PerCP-CD45 (CD45 as a lineagemarker) or a cocktail of labeled CD2, 3, 10, 16, 18, 31, 45, 64, 140bantibodies and 7AAD (to exclude dead cells) for 30 min. After 2 washes,cells were analyzed by flow cytometry. The same staining procedureapplies to breast tumor cell lines, except the use of PerCP-CD45 (CD45as a lineage marker) or a cocktail of labeled CD2, 3, 10, 16, 18, 31,45, 64, 140b antibodies.

Immunohistochemistry

CSPG4: Formalin-fixed paraffin-embedded (FFPE) sections of tumorsobtained from patients were deparaffinized and hydrated. Antigenretrieval was performed by boiling for 15 mins in 1 mM EDTA (pH 8.0).Slides were blocked by 3% hydrogen peroxidase and 1% bovine serumalbumin/5% normal horse serum in Tris-buffered saline with Tween 20(Sigma-Aldrich Inc., St. Louis, Mo.), and incubated overnight at 4° C.in a closed humid chamber with the CSPG4-specific mAb, D2.8.5-C4B8 (3μg/ml). Signals were amplified with EnVision+System-HRP (DakoCytomationInc) and developed by diaminobenzidine (DAB, DakoCytomation Inc.).Samples were counterstained with hematoxylin, dehydrated, and mounted inCanada balsam (Sigma-Aldrich Inc.).

Breast cancer sections stained with CSPG4-specific mAb were gradedseparately by two investigators utilizing the modified protocol aspreviously described (Kageshita, T., et al., Cancer Res 53, 3349-3354,1993).

CD31: Zinc (Zinc Fixative, BD Pharmingen) fixed and paraffin embeddedtissue sections of surgical removed xenografts were stained withanti-mouse CD31(PECAM1) antibody as previously described (Ko et al.,Cancer Res 67, 7875-7884, 2007).

Phopos-Histone 3:

FFPE of lung sections were stained with anti-p-Histone3 antibodyaccording to the manufacture's protocol.

Apoptosis:

FFPE of lung sections were stained for apoptotic cells using APOP* PLUSPEROXIDASE IN SITU APOPTOSIS KIT™ (S7101) (TerminalDeoxynucleotidyltransferase-Mediated dUTP Nick End Lebeling (TNUEL)assay) (Millipore) according to the manufacture's protocol.

RT-PCR.

RT-PCR was performed as described (Luo, et al., Oncogene 25, 2873-2884,2006).

Western Blotting.

Western blot assay for CSPG4 was performed as described (Wang, X., etal. J Immunol Methods 299, 139-151, 2005); for other signaling relatedproteins were performed using standard techniques on the following twotypes of cell lysates: 1) lysate of cultured cells (2×10⁴ cells/well ina 96-well plate were serum starved for 48 hours, then treated witheither mAb 225.28 (0.1 mg/ml), isotype (0.1 mg/ml) or PBS for additional48 hours), cells were lysed in lysis buffer (10 mM Tris-HC, 1% NP40, 1mM EDTA, 0.1% BSA, 150 mM NaCl, 1/50 of protease inhibitor cocktail(Calbiochem) and 2) lysate of snap frozen surgically removed xenografts,tissues were homogenized before and after adding an ice-cold RIPA buffer(Thermo Scientific) containing 1/50 of protease inhibitor cocktail(Calbiochem). After vortex 60 seconds, the samples were ice-cooled for45 min Insoluble material was removed from tissue lysates bycentrifugation at 13,000 rpm for 30 mM at 4° C. Protein concentrationwas measured by Bradford reagent (BioRad).

Cell Growth, Adhesion and Migration.

For cell growth assay, cells (5×10⁴ cells/well) were serum starved 48hours and then seeded in a 96-well plate containing 4 times dilutedmatrigel (growth factor-reduced matrigel-CB-40230, BD Biosciences) and0.25 mg/ml either mAb 225.28, control mAb F3C25 or PBS in serum freeRPMI 1640 medium (total volume 200 μl/well) and cultured in a 37° C. and5% CO₂ incubator for 6 days. Then pictures of cells were taken and cellswere trypsinized and counted. For the cell adhesion assay, cells (2×10⁵cells/well) were seeded and incubated with 0.05 mg/ml either mAb 225.28,control mAb F3C25 or PBS for 40 minutes in a 96-well plate which wascoated with 100 μl/well of 12 μg/ml fibronectin in PBS. Non-adherentcells were washed away with PBS. Adherent cells were fixed by 70%ethanol, stained by crystal violet and resuspended in PBS. Absorbance at540 nm was measured. The results were expressed as % inhibition ofadhesion, utilizing the adhesion values obtained in PBS without mAb as areference. The values shown were the mean of three independentexperiments. For the migration assay, cells (5×10⁴ cells/well) wereserum starved 48 hours and then seeded in a 24-transwell plate (24-wellinsert, pore size 8 μm; BD Biosciences) with 0.25 mg/ml mAb 225.28,control mAb F3C25 or PBS. The cells migrated toward to serum-freeRPMI1640 medium containing 10 μg/ml fibronectin. After 48 hours,migrated cells were stained with HEMA 3 stain set, taken picture andcounted under a Zeiss Inverted Fluorescence Microscope (AxioVisionSoftware). Mean of six independent high power field (100×) are shown ascolumns All of the above experiments were performed in triplicate.

Experimental Lung Metastasis and Treatment Experiments.

SCID mice were intravenously injected with cells MDA-MB-435 orMDA-MB-231 and treated with mAb, as indicated in description of FIG. 4.

Spontaneous Lung Metastasis and Treatment Experiments.

MDA-MB-435 cells were implanted into mammary fat pad of SCID mice.Primary tumors were surgically removed and mAb was administered asindicated in the description of FIG. 5.

Example 2 Preferential CSPG4 Expression in ER Negative and Her2 NegativeBreast Cancers

Comparison of the CSPG4 mRNA levels in ER⁻/Her2⁻, ER⁻/Her2⁺, ER⁺/Her2⁻,and ER⁺/Her2⁺ breast cancer cells using a publicly available, clinicallyannotated breast cancer data set (GSE5460) showed that the CSPG4 geneexpression level was significantly (Mann Whitney, p=0.01 to 0.06) higherin the ER⁻/Her2⁻ subgroup than in the other subgroups. It appeared to bebest correlated with the basal breast cancer phenotype (ER−/Her2−) (FIG.1 a).

The preferential CSPG4 expression by ER⁻, PR⁻ and Her2⁻ triple negativebreast cancer tissues was corroborated by the results ofimmunohistochemical staining the in different subtypes of human breastcancer using CSPG4-specific mAb. CSPG4 was detected in 72.7% of triplenegative breast cancer tissue from 44 patients. In contrast, CSPG4 wasdetected in 28.6% in ER⁺ breast cancer tissue from 18 patients and 16.7%of 28 ER+ primary breast cancer lesions an 18 Her2⁺ breast cancer tissuefrom 18 patients, respectively (Table 1 and FIG. 1 b).

TABLE 1 Differential expression of CSPG4 on tumor subtypes CSPG4⁺ ER⁺Her2⁺ ER⁻PR⁻Her2⁻ (−) 20/28 (71.4%) 15/18 (83.3%) 12/44 (27.3%) (+) 6/28(21.4%) 3/18 (16.7%) 10/44 (22.7%) (++) 2/28 (7.2%) 0/18 (0%) 18/44(40.9%) (+++) 0/28 (0%) 0/18 (0%) 4/44 (9.1%)The grading system used is as described in description for FIG. 1 b.

Example 3 CSPG4 Expression by a Subpopulation of Breast CSC in TNBC CellLines

The frequency of CSC, identified as CD44⁺CD24^(−/lo) cells by flowcytometry, is much higher in TMBC than in luminal breast cancer celllines. They were not detectable in the cell line SK-BR-3, and had afrequency of 1.5 and 34.6% in the cell lines T-47D and MCF-7,respectively. In contrast, they had a frequency of 92.5-99.0% in theTNBC cell lines HS578T, MDA-MB-231, MDA-MB-435 and SUM-149¹² (FIG. 1 c).Interestingly, CSPG4 is differentially expressed on CD44⁺ CD24^(−/lo)cells present in basal and luminal breast cancer cell lines. It has ahigh expression on a high percentage (66.7-96.1%) of CSC in the 4 TNBCcell lines, but only a low or barely detectable expression on a lowpercentage (1.5-13.0%) of CSC in the three luminal breast cancer celllines. (FIG. 1 c).

Flow cytometric analysis showed that the distinct determinantsrecognized by the CSPG4-specific mAb 225.28, 763.74, TP41.2 and TP61.5are differentially expressed on each cell line, in terms of percentageof stained cells and of staining intensity. In the SUM-149 cell lineabout 95% of CSC express the determinant recognized by mAb 225.28 andless than 5% those recognized by mAb 763.74 and TP41.2. In contrast, inthe MDA-MB-435 cell line at least 90% of CSC express the determinantsrecognized by the four CSPG4-specific mAbs. The expression patterns ofCSPG4 determinants on whole cell populations are comparable to those onautologous CSC in the MDA-MB-435 and SUM-149 cell lines. In contrast, inthe MDA-MB-435 cell line about 90% of CSC express the determinantsrecognized by the four CSPG4-specific mAbs. The expression patterns ofthe CpSG4 determinants recognized by mAb 763.74 and TP41.2 is lower onCSC than on the autologous whole cell populations in the HS578T andMDA-MB-231 cell lines, suggesting a unique modification of CSPG4epitopes expressed on CSC (Table 2a, 2b*).

TABLE 2a Differential expression of distinct CSPG4 determinants on wholecell population of breast cancer cell lines MDA- MDA- SK- HS578T MCF-7MB-231 MB-435 BR-3 SUM-149 T-47D mAb %/MFI %/MFI %/MFI %/MFI %/MFI %/MFI%/MFI 225.28 66.7/379.2  8.5/12.0 83.8/53.8  96.1/713.6 1.5/4.6 93.4/193.1 13.0/3.8  763.74 61.4/39.2  17.4/13.8  91.2/154.1 93.5/79.20.0/2.0 1.3/2.8 0.5/3.4 TP41.2 52.6/34.2  13.8/11.9  88.4/142.386.8/63.3 0.1/2.1 0.9/2.8 0.3/2.7 TP61.5 92.3/109.4 3.5/7.8 68.3/86.382.4/61.1 0.1/2.4 51.1/19.6 0.0/2.3 Isotype 0.4/13.0 0.7/6.2 0.3/9.80.7/9.1 1.9/4.3 2.0/2.7  6.2/10.4 control

TABLE 2b Differential expression of distinct CSPG4 determinants onputative CSC in breast cancer cell lines MDA- MDA- SK- HS578T MCF-7MB-231 MB-435 BR-3 SUM-149 T-47D mAb %/MFI %/MFI %/MFI %/MFI %/MFI %/MFI%/MFI 225.28  71.7/195.8 11.4/9.8  85.7/53.8  99.7/694.4 NA  95.7/179.0NA 763.74 33.9/17.6 2.6/2.4 16.6/6.9  96.4/50.5 NA 3.0/3.1 NA TP41.217.6/14.5 2.5/2.5 12.0/6.1  91.9/40.4 NA 1.4/2.7 NA TP61.5 72.4/34.49.9/3.2 75.6/33.6 98.0/93.0 NA 51.2/10.5 NA Isotype  0.5/10.0 0.6/1.80.4/3.7 0.5/3.0 NA 0.5/2.5 NA control *For the data shown in the tables,the he TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435 and SUM-149 andthe luminal cancer cell lines MCF-7, SK-BR-3 and T-47D were stained withthe mAb 225.28, 763.74, TP41.2 and TP61.5 which recognize distinct CSPG4determinants and subjected to FACS analysis. The tables indicate the %of stained cells and the mean fluorescence intensity (MFI) (a) in thewhole cell population and (b) in the subpopulation of putative CSC,defined as CD44⁺/CD24^(−/lo). 7AAD⁺ cells (dead cells) were excludedthrough gating in both experiments. NA, not applicable.

The heterogeneity in the expression of CDPG4 determinants is likely toreflect the differences in the glycosylation of this molecule.CSPG4-specific mAb showed only minor variations across multiple cellculture passages. These results indicate that the expression of CSPG4 bya highly tumorigenic subpopulation of breast cancer cells is a stablecharacteristic.

Example 4 CSPG4 Expression by a Subpopulation of Breast CSC in PleuralEffusions from Patients with Breast Cancer

Utilizing the method of Al-Hajj et al. (Proc Natl Acad Sci USA100:3983-3988), 0.71-92.7% of breast CSC were identified aslineage-negative (CD2, 3, 10, 16, 18, 31, 45, 64, 140b), CD45⁻, CD44⁺,CD24^(−/lo) in cells isolated from pleural effusions of 14 breast cancerpatients (Table 2). Cells with this surface phenotype have been shown tobe enriched for highly tumorigenic subpopulations. The percentage of CSCin the lineage-negative pleural effusion cells was low (<10%) in ninepatients, intermediate (18.6-35.0%) in four patients and high (92.7%) inone patient. Consistent with the CSC phenotype, the percentage of CSC inwhole effusion cells ranged from 0.01% to 0.89% (Table 2). The extent ofenrichment differed depending on the CSPG4-specific mAb used forstaining, a result consistent with the heterogeneity in the expressionof the corresponding determinants observed on cell lines (Table 3).

TABLE 3 Enrichment of putative CSC population in CSPG4 positive cells inpleural effusions from patients with breast carcinoma* CD44⁺CD24^(−/lo)cells in % of CD44⁺CD24^(−/lo) in CSPG4⁺ cells (fold enrichment) Highest% Patient CD45⁻ cells mAb mAb mAb mAb and fold sample # (%) 225.28763.74 TP41.2 TP61.5 Average enrichment PC-P4 2.91 8.6 (2.96)  5.7(1.96) 10.2 (3.51) 24.7 (8.49) 12.3 (4.23) 24.7 (8.49) PC-P5 16.3 26.8(1.64) 68.9 (4.23) 23.7 (1.45) 18.3 (1.12) 34.4 (2.11) 68.9 (4.23) PC-P67.21 4.7 (0.65)  0.0 (0.00) 3.57 (0.50) 50.0 (6.93) 14.6 (2.02) 50.0(6.93) PC-P7 19.2 35.5 (1.85) 95.4 (4.97) 61.3 (3.19) 75.4 (3.93) 66.9(3.48) 95.4 (4.97) PC-P8 18.0 2.9 (0.16) 60.7 (3.37) 20.9 (1.16) 31.3(1.74) 29.0 (1.61) 60.7 (3.37) PC-P9 3.38 22.6 (6.69) 40.2 (11.9) 38.5(11.4) 35.7 (10.6) 34.3 (10.1) 40.2 (11.9) PC-P10 31.6 91.5 (2.90) 96.8(3.06) 93.7 (2.97) 94.8 (3.00) 94.2 (2.98) 96.8 (3.06) PC-P11 13.0 67.4(5.18) 93.3 (7.18) 70.3 (5.41) 75.5 (5.81) 76.6 (5.89) 93.3 (7.18)PC-P12 4.94 13.5 (2.73) 96.2 (19.5) 90.3 (18.3) 67.3 (13.6) 66.8 (13.5)96.2 (19.5) PC-P13 11.6 71.0 (6.12) 81.4 (7.02) 68.7 (5.92) 76.7 (6.61)74.5 (6.42) 81.4 (7.02) PC-P14 12.2 8.4 (0.69) 91.5 (7.50) 49.1 (4.02)32.0 (2.62) 45.3 (3.71) 91.5 (7.50) PC-P15 58.7 69.3 (1.18) 90.3 (1.54)ND ND 79.8 (1.36) 90.3 (1.54) Average 16.59 35.2 (2.73) 68.4 (6.02) 48.2(5.25) 52.9 (5.86) 52.4 (4.78) 74.1 (7.14) (fold enrichment) *Tumorcells from pleural effusions from 12 patients with metastatic breastcancer were sequentially incubated with CSPG4-specific mAb 225.28,763.74, TP41.2, or TP61.5, with PE-labeled anti-mouse IgG antibodies andwith FITC-labeled anti-CD24, APC-labeled anti-CD44, PerCP-labeledanti-CD45, and 7-AAD. Stained cells were subjected to FACS analysis. Thepercentages of CD44⁺CD24^(−/lo) cells in the CD45⁻ 7-AAD⁻ population andin the CD45⁻ 7-AAD⁻ CSPG4⁺ population were determined. Enrichment ofCD44⁺CD24^(−/lo) population by gating at CSPG4 positive cells wascalculated by dividing the percentage of CD44⁺CD24^(−/lo) cells in theCD45⁻ 7-AAD⁻ CSPG4⁺ population by that in the CD45⁻ 7-AAD⁻ populationand is shown in parenthesis in each well. The highest percentages andfold enrichment are shown in the right column for each patient's sample.

TABLE 4 Percentage of putative CSC in pleural effusions from patientswith breast carcinoma Total CD44⁺CD24^(−/lo) CD44⁺CD24^(−/lo) Patientcell cells in cells in CD44⁺CD24^(−/lo) sample number CD45⁻Lin⁻ totalcell number # (×10⁶) cells (%) cells (%) (×10³) PC-P1 34 0.71 <0.01 0.8PC-P2 40 18.60 0.01 4.0 PC-P3 6 8.57 0.08 4.8 PC-P4 280 1.61 0.01 28.0PC-P5 4170 2.53 0.34 14178.0 PC-P6 298 35.00 0.89 2652.2 PC-P7 220 28.900.01 22.0 PC-P8 360 23.10 0.01 36.0 PC-P9 98 1.56 <0.01 9.6 PC-P10 13007.14 <0.01 4.5 PC-P11 200 4.01 0.01 20.0 PC-P12 1000 1.64 <0.01 1.6PC-P13 2515 0.99 <0.01 12.4 PC-P15 58 92.70 0.07 40.6

Example 5 Molecular Profile of CSPG4 Expressed by Breast CSC

CSPG4 mRNA was detected by RT-PCR in MDA-MB-435 cells, most (99%) ofwhich express the CSC phenotype (CD44⁺CD24^(−/lo)) (FIG. 6 a). Westernblot analysis with mAb 763.74 of a MDA-MB-435 cell lysate detected thetwo components of CSPG4 (FIG. 6 b).

Example 6 Inhibition by CSPG4-Specific mAb of Tumor Cell Growth,Adhesion and Migration In Vitro via Inhibiting PI3K/PTEN/Akt and MARPKPathways Signaling

Previous work had shown that CSPG4 promotes progenitor and tumor cellmotility, adhesion and growth resulting in melanoma and glioma cellgrowth and metastasis. The CSPG-specific monoclonal antibodies weretested for their ability to block tumor cell motility and growth invitro. As shown in FIG. 2, CSPG4 specific mAb 225.28 inhibited cellgrowth, adhesion and migration in CSPG4⁺ TNBC cells in vitro. As shownin FIG. 2, TNBC cells in the presence of mAb 225.28 at indicated dosagesexhibited 70% inhibition (FIG. 2 a,b) of cell growth in a 3D matrixsetting, which resembles closely to in vivo tumor growth conditions; theadhesion of these cells to fibronectin was also inhibited by 45% (FIG. 2c) and the motility of these cells towards fibronectin in a Boydenchamber assay was inhibited by 56% (FIG. 2 d).

Previous studies had shown that CSPG4 functions to modify multiplesignal transduction pathways that impact on the cytoskeleton, growth,motility and survival of tumor cells. Specifically, several key pathwayshave been identified as related to the function of CSPG4. These pathwaysinclude protein kinase C(PKC) α, FAK, Erk 1, 2 and PI3 kinase, which areall activated and associated with triple negative breast cancer. Asshown in FIG. 3, mAb 225.28 in vitro incubation with the CSPG4-specificmonoclonal antibody 225.28 inhibited the expression of PKCα and well asthe activation of FAK, Erk 1, 2 and AKT in both MDA-MB-231 andMDA-MB-435 cells.

Example 7 Inhibition/Regression by CSPG4-Specific mAb of Human TNBCEstablished Experimental Metastasis In Vivo

The CSPG4-specific mAb 225.28 inhibited experimental metastases ofMDA-MB-231 cells, which have a high percentage of CD441CD24^(−/lo).cells and a medium to high level of CSPG4 expression (FIG. 4A). On day 3following an intravenous (i.v.) injection of tumor cells, mice weredivided randomly into two groups: one group was injected twice weeklywith mAb 225.28 (100 μg/injection) and the other one with a control mAb(100 μg/injection). Evaluation of mice 79 days post tumor cellinoculation demonstrated that CSPG4-specific mAb inhibited MDA-MB-231metastasis by greater than 99% compared to the control mAb. Similarly,metastasis of MDA-MB-435 cells, which have a high CSPG4 expression, anda CD44₊/CD24_(−/lo) phenotype similar to that of MDA-MB-231 cells, wasinhibited by greater than 95% with either mAb 225.28 or mAb 763.74compared to control mAb (FIG. 4 b).

Example 8 Inhibition by CSPG4-Specific mAb of Human Orthotopic TNBCTumor Post-Surgery Recurrence and Spontaneous Metastasis In Vivo viaInhibiting PI3K/PTEN/Akt and MARPK Pathways Signaling

To conduct a study which is clinical relevant, the ability ofCSPG4-specific mAb to inhibit tumor recurrence and spontaneousmetastasis of human breast MDA-MB-435 mammary tumors in SCID mice wasexamined following surgical removal of primary tumors, a setting whichresembles the human disease clinically. Mice treated with mAb 225.28 and763.74 had significantly lower spontaneous lung metastases (FIG. 5 a)than those administered with the control mAb. In addition, only onesmall size local tumor recurrence and no recurrences were detected inthe two groups of 5 mice each, treated with mAb 763.74 and 225.28,respectively. In contrast, three large size tumor recurrences were foundin the five mice treated with the control mAb (FIG. 5B). The results arerepresentative of two independent experiments. The results indicate thattargeting CSPG4 can be useful clinically to inhibit both tumormetastasis and recurrence after primary tumor surgical removal.

Primary tumors surgically removed from mice treated with mAb 225.28exhibited a significantly lower level of vascular density compared toprimary tumors from mice treated with a control mAb (FIGS. 5C and 5D).The effect on vascular density may be due in part to the targeting ofpericytes which express CSPG4 and are important for the maturation ofnewly forming vessels within tumors.

Cell lysates generated from primary tumors removed from mice treatedwith the CSPG4-specific mAb 225.28 and from mice treated with a controlmAb were also evaluated for the activation of specific signaltransduction pathways associated with TNBC growth and progression. Theyinclude the integrin mediated signaling, as shown by FAK activation andthe key tumor cell-growth promoting and -survival signaling pathways, asshown by activation of Erk 1,2 and Akt, respectively. The activation ofthese three signaling pathways was markedly reduced in the primarytumors removed from the mice treated with the CSPG4-specific mAb 225.28as compared to those removed from the mice treated with a control mAb.Furthermore, primary tumors removed from the mice treated withCSPG4-specific mAb also exhibited lower levels of PKCα compared to thosefrom the mice treated with a control mAb (FIG. 5E). These results showthat targeting CSPG4 with mAb has significant inhibitory effects on thecell growth, adhesion and survival related pathways important for breastcancer progression and metastasis.

Gene-expression profiling, immunochemical studies andimmunohistochemical staining have convincingly shown that in breastcancer CSPG4 is preferentially expressed in basal/TNBC. The differentialexpression of CSPG4 in basal and luminal breast cancer both indifferentiated cells and in cells with the CSC phenotype is not uniqueto CSPG4, since an association between EGFR expression and thebasal-like phenotype has also been demonstrated (Korshing et al., LabInvest 82:1525-1533, 2002). Furthermore, IHC staining has detectedcytokeratins CK5/6, S-phase kinase-associated protein 2 (SKP2) andmesenchyme forkhead 1 (FOXC2) mainly in basal/TNBC lesions (Mani et al.,Proc Natl Acad Sci USA 104:10069-10074, 2007). Whether the differentialCSPG4 expression in TNBC and other breast cancer subtypes reflects thedifferent cell types from which they originate, or differences in themechanism(s) of regulating gene expression in different subtypes ofbreast cancer remains to be determined. On the other hand the lack orlow level of CSPG4 expression in other subtypes of breast cancer is notlikely to reflect loss of the encoding genes, since loss of geneticmaterial in chromosome 15 where CSGP4 has been mapped (28) has not beendescribed in these subtypes of breast cancer. Whatever the molecularmechanism(s) underlying CSPG4 gene activation in TNBC is (are), itspreferential expression in this breast cancer subtype suggests itspotential use as a diagnostic biomarker and as a therapeutic target.

In TNBC, CSPG4 appears to be expressed mostly, although not exclusivelyon subpopulations with the CD44⁺CD24^(−/lo). phenotype. This cellsubpopulation, which is enriched in this subtype of breast cancer,appears to play a major role in progression, since cells with aCD44⁺CD24^(−/lo). phenotype have a high invasive potential thatfacilitates metastasis to lung (Sheridan, Breast Cancer Res 8:R59, 2006)and represent the majority of cancer cells detected in the bone marrowof breast cancer patients (Balic et al., Clin Cancer Res 12:5615-5621,2006). Given the role of CSPG4 in migration, metastasis (Burg et al, JCell Physiol 177:299-312, 1998) and chemo-resistance (Checkenya,Oncogene 27:5182-5194, 2008) of malignant cells, as well as itsexpression on pericytes (Ozerdem, Angiogenesis 7:269-276, 2004), whichmay be the origin of mesenchymal cells (Crisen, Cell Stem Cell3:301-313, 2008), it is possible that that the core protein of this cellsurface proteoglycan contributes to the stem-like properties of thissubpopulation and to their epithelial-mesenchymal transition (EMT). Thelatter morphologic transdifferentiation process enables carcinoma cellsto acquire a mesenchymal appearance/gene expression profile whichcontributes to increased motility and invasiveness during malignantprogression (Hay, Acta Anat (Basel) 154:8-20, 1995).

CSPG4-specific mAb are effective at inhibiting the metastasis ofmultiple TNBC cell lines, including MDA-MB-231 and MDA-MB-435 cells. TheMDA-MB-435 cell line is tested in these studies as a model forevaluating the tumor metastasis and recurrence of CSPG4⁺CD44⁺CD24^(−/lo)cells and for establishing proof of principle for the efficacy oftargeting CSPG4 with mAb in cells with aggressive metastatic phonotypein a preclinical model.

Basal/TNBC are associated particularly with aggressive behavior and poorprognosis, and typically do not express hormone receptors or HER-2 (the“triple-negative” phenotype). Therefore, TNBC patients are unlikely tobenefit from currently available targeted systemic therapy. Moreover,EGFR targeted therapy by cetuximab in metastatic basal/TNBC yieldedrather disappointing results in clinical trials. To overcome thislimitation, proteins predominantly expressed on basal/TNBC, such asCSPG4, are attractive candidates for therapeutic targets. Thefunction-blocking CSPG4-specific mAb 225.28, inhibits in vitro tumorcell growth, adhesion and migration and in vivo tumor cell proliferationand tumor angiogenesis and induce tumor cell apoptosis within theprimary tumor consisting of TNBC cells. As a result, CSPG4-specific mAbcaused >70% regression of established TNBC cell derived lung metastasesand inhibited recurrences and metastatic spreading following thesurgical removal of primary tumors.

Basal/TNBC are associated particularly with aggressive behavior and poorprognosis, and typically do not express hormone receptors or HER-2 (the“triple-negative” phenotype). Therefore, TNBC patients are unlikely tobenefit from currently available targeted systemic therapy. Moreover,EGFR targeted therapy by cetuximab in metastatic basal/TNBC has yieldedrather disappointing results in clinical trials within the primary tumorconsisting of TNBC cells. As a result, CSPG4-specific mAb causes >70%regression of established TNBC cell derived lung metastases and inhibitsrecurrence and metastasis of primary tumors that have been removedsurgically. CSPG4-specific mAb also inhibit the activation of signaltransduction pathways important for the malignant progression of TNBCcells.

Previous studies have demonstrated that CSPG4 and the NG2 rat homologueare associated with several of these key pathways in other tumor models.These include those important for cytoskeletal reorganization (e.g.cdc42 and Rac), integrin mediated adhesion (e.g. FAK activation), growthpromoting pathways (e.g. sustained Erk 1,2 activation) and cell survivalpathways (e.g. Akt activation). Targeting CSPG4 with mAb has apleiotropic effect on the TNBC cells. This is in contrast to othercurrent therapeutic mAb such as herceptin, which appear to targetPI3K/Akt related pathways without inhibiting growth related signalingpathways such as the sustained activation of Erk 1,2. The CSPG4-specificmAb 225.28 did not appear to exhibit antibody-dependent, cell-mediatedcytotoxicity as in vivo depletion of NK cells in SCID mice had no impacton its therapeutic efficacy. Furthermore, the antibody did not mediatecomplement-dependent cytotoxicity of tumor cells. Collectively, thesedata indicate that the inhibitory effect of mAb 225.28 on tumor growthand metastasis is a direct result of its ability to inhibit signalingpathways important for the malignant progression of tumor cells.

There are significant efforts to target certain proteins, such as SHhand wnt related signaling pathways that are associated with a ‘stem celllike’ tumor phenotype. CSPG4 has restricted distribution in normaltissues and is associated with progenitor populations within differentorgans. However, it offers advantages as a therapeutic target over othertargets as the SHh and Wnt signaling pathways, since CSPG is notfundamentally required for normal stem cell maintenance and selfrenewal. Consequently, targeting of CSPG4 with mAb is unlikely to beassociated with the negative effects on normal tissue stem cells.CSPG4-specific immunity does not appear to cause side effects in humansand did not cause general toxicity, such as body weight loss and delayin wound healing in mice after a half year systemic administration(twice weekly) of mAb 225.28. The exquisite specificity ofCSPG4-specific mAb makes these antibodies ideally suited for targetinghighly tumorigenic/metastatic cell subpopulations within malignanttumors. It is disclosed herein that the exquisite specificity ofmonoclonal antibodies may be ideally suited to treatment of cancer stemcells bearing high levels of CSPG4 expression.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

The invention claimed is:
 1. A method for inhibiting the growth of atriple negative basal breast cancer cell in a subject, comprising:selecting a subject that has triple negative basal breast cancer; andcontacting the triple negative basal breast cancer cell with aneffective amount of an antibody that specifically binds chondroitinsulfate proteoglycan 4 (CSPG4), thereby inhibiting the growth of thetriple negative cancer cell.
 2. The method of claim 1, wherein theantibody is covalently linked to an effector molecule.
 3. The method ofclaim 1, wherein the antibody is a monoclonal antibody.
 4. The method ofclaim 2, wherein the effector molecule is a chemotherapeutic agent. 5.The method of claim 2, wherein the effector molecule comprises a toxicmoiety.
 6. The method of claim 5, wherein the toxic moiety is selectedfrom the group consisting of ricin A, abrin, diphtheria toxin or asubunit thereof, Pseudomonas exotoxin or a portion thereof, andbotulinum toxins A through F.
 7. The method of claim 6, wherein thePseudomonas exotoxin is selected from the group consisting of PE35,PE37, PE38, and PE40.
 8. The method of claim 1, wherein inhibiting thegrowth of the basal breast cancer cell comprises reducing metastasis ofthe basal breast cancer cell in the subject.
 9. The method of claim 1,wherein the subject has undergone surgical removal of a triple negativebasal breast carcinoma primary tumor; and wherein inhibiting the growthof the basal breast cancer cell comprises preventing tumor recurrence invivo.
 10. The method of claim 1, further comprising administering to thesubject an additional chemotherapeutic agent.
 11. The method of claim10, wherein the additional chemotherapeutic agent is a mitoticinhibitor, an alkylating agent, an anti-metabolite, an intercalatingantibiotic, a growth factor inhibitor, a cell cycle inhibitor, anenzyme, a topoisomerase inhibitor, an anti-androgen, or ananti-angiogenesis agent.
 12. The method of claim 1, wherein the methodcomprises selecting a subject with metastatic triple negative basalbreast cancer.